Spinal cord from body donors is suitable for multicolor immunofluorescence

Vol.:(0123456789) 1 3 Histochemistry and Cell Biology https://doi.org/10.1007/s00418-022-02154-5 ORIGINAL PAPER Spinal cord frombody donors issuitable formulticolor immunofluorescence LukasF.Reissig1· GenovaCarrero‑Rojas1· UdoMaierhofer2· AtiehSeyedianMoghaddam1· AndreasHainfellner1· BernhardGesslbauer2· ThomasHaider3· JohannesStreicher4· OskarC.Aszmann2· AngelM.Pastor5· WolfgangJ.Weninger1· RolandBlumer1 Accepted: 10 September 2022 © The Author(s) 2022 Abstract Immunohistochemistry is a powerful tool for studying neuronal tissue from humans at the molecular level. Obtaining fresh neuronal tissue from human organ donors is difficult and sometimes impossible. In anatomical body donations, neuronal tissue is dedicated to research purposes and because of its easier availability, it may be an alternative source for research. In this study, we harvested spinal cord from a single organ donor 2h (h) postmortem and spinal cord from body donors 24, 48, and 72h postmortem and tested how long after death, valid multi-color immunofluorescence or horseradish peroxidase (HRP) immunohistochemistry is possible. We used general and specific neuronal markers and glial markers for immunolabeling experiments. Here we showed that it is possible to visualize molecularly different neuronal elements with high precision in the body donor spinal cord 24h postmortem and the quality of the image data was comparable to those from the fresh organ donor spinal cord. High-contrast multicolor images of the 24-h spinal cords allowed accurate automated quantification of different neuronal elements in the same sample. Although there was antibody-specific signal reduction over postmortem intervals, the signal quality for most antibodies was acceptable at 48h but no longer at 72h postmortem. In conclusion, our study has defined a postmortem time window of more than 24h during which valid immunohistochemical information can be obtained from the body donor spinal cord. Due to the easier availability, neuronal tissue from body donors is an alternative source for basic and clinical research. Keywords Human spinal cord· Postmortem interval· Multicolor immunofluorescence· Horseradish peroxidase immunohistochemistry· Neuronal markers Introduction Immunohistochemistry is a powerful tool for studying neuronal tissue from humans at the molecular level and this is the foundation for a better understanding of neuronal function. Typically, human neuronal tissue is obtained postmortem from organ donors or body donors who donated their bodies to anatomical departments. The use of fresh neuronal tissue from organ donors for research is limited and due to ethical reasons often prohibited. In contrast, tissue from body donors can be obtained much easier because body donors have already given written consent during a lifetime that his/her body is dedicated to teaching and scientific purposes. For this reason, neuronal tissue from body donors should be tested as an alternative source for immunohistochemical studies. * Roland Blumer [email protected] 1 Division ofAnatomy, MIC, Medical University Vienna, Vienna, Austria 2 Clinical Laboratory forBionic Extremity Reconstruction, Department ofPlastic, Reconstructive andAesthetic Surgery, Medical University ofVienna, Vienna, Austria 3 Department ofOrthopedic andTrauma Surgery, Medical University ofVienna, Vienna, Austria 4 Department ofAnatomy andBiomechanics, Division ofAnatomy andDevelopmental Biology, Karl Landsteiner University ofHealth Science, KremsanderDonau, Austria 5 Departamento de Fisiología, Facultad de Biología, Universidad de Sevilla, Seville, Spain Histochemistry and Cell Biology 1 3 A prerequisite for immunohistochemical analyses is optimal tissue quality. To avoid loss of quality, it is recommended to refrigerate bodies after death and keep the time between death and the start of tissue fixation (postmortem interval) as short as possible (Werner etal. 2000; Waldvogel etal. 2006; Almulhim and Menezes 2022). However, such a controlled procedure is very challenging, and only possible for donors who die in an intensive care unit. People who donate their bodies to anatomical departments often die outside a monitoring facility and as there is uncertainty about the exact time of death, inaccuracies, and variability in postmortem interval estimates are unavoidable. Additionally, there is uncertainty about the ambient temperature bodies are exposed to until they are transferred to anatomical departments. Although neuronal tissue (peripheral nerves) from body donors has been analyzed by immunohistochemistry (Rein etal. 2013, 2015, 2020; Blumer etal. 2016; Pascual-Font etal. 2016; Garcia-Mesa etal. 2017; Lienbacher etal. 2019), it would be crucial to define a period after death during which accurate immunohistochemistry is possible in body donors. Such information is currently lacking but would be relevant for scientists who analyze nerve tissue from body donors at the molecular level. In the present study, we harvested spinal cord tissue samples from body donors as they routinely arrive at body donor facilities at 24, 48, and 72h postmortem intervals. We used a series of general and specific neuronal markers as well as glial markers and evaluated the stability of multi-color immunofluorescence and horseradish peroxidase (HRP) immunohistochemistry. Results in body donor spinal cords were compared with immunolabeling assays in fresh spinal cord obtained 2h postmortem from an organ donor. We demonstrate that 24h after death, different neuronal elements can be identified with high fidelity in the human spinal cord by immunofluorescence/HRP immunohistochemistry and high contrast images can be used for accurate computerbased quantitative analyses. With prolonged postmortem intervals (48 and 72h), the staining capacity of the antibodies gradually decreases in an antibody-specific fashion. Materials andmethods Tissue collection andpreparation From 15 body donors, the cervical segments 4–6 of the spinal cord were collected and analyzed. Spinal cords were from voluntary body donors of both sexes (seven females and eight males) who provided written consent to donate their dead bodies to be used for teaching and research at the Center of Anatomy and Cell Biology, Medical University of Vienna. The age of the donors ranged between 58 and 103years (mean and standard deviation 81.2 ± 13.08) and none of them had a history of neurological disorders. Once delivered to the Department of Anatomy, body donors were kept in a mortuary at 4°C until dissection. For all body donors, the time of death was precisely known, and they were divided into three groups. Each group contained five subjects. In the first group, the time between death and start of tissue fixation (postmortem interval) was 24h, in the second 48h, and in the third group 72h. Following dissection, the cervical spinal cord was subdivided into three pieces, each piece corresponding to a single spinal cord segment. Then, the issue was immersion fixed by 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB, pH 7.4) for 24h at 4°C and afterward stored in phosphatebuffered saline (PBS) for further processing. In every group, handling procedures for tissue processing were the same. To evaluate results in the spinal cord from body donors, we analyzed the spinal cord (thoracic segments) of an organ donor (39 years old) used in a previous study (Gesslbauer etal., 2017). The postmortem interval of the organ donor spinal cord was 2h and because of this short postmortem interval, it was used as a control for all other studied groups. The study has been approved by the local ethics committee (protocol number of the approval No. 1213/2012 and 1373/2021). Tissue preparation andprocessing Spinal cords from the organ donor and body donors were cryoprotected in graded concentrations of sucrose (10, 25, and 40%) in PBS containing 0.05% sodium azide to avoid bacterial and fungal contamination, frozen, and stored at −80°C according to our guidelines for cryo-embedding (Blumer etal. 2019). For histological and immunohistochemical evaluation, tissue was cryo-sectioned at 10-μm thickness. Histology Human spinal cord sections were labeled with hematoxylin–eosin using a standard protocol (hematoxylin 3min and eosin 1min). Thereafter, sections were dehydrated in graded solutions of alcohol and coverslipped in DPX mounting medium (Merck/Millipore, Temecula, CA USA). Immunohistochemistry Antibodies andantibody characterization Different neuronal marker antibodies were used to visualize nervous elements in the spinal cord. This included panneuronal markers (anti-neurofilament, anti-synaptophy- sin, and anti-nuclear neuronal protein), specific neuronal markers (anti-calcitonin gene-related peptide, anti-choline Histochemistry and Cell Biology 1 3 acetyltransferase, and anti-vesicular acetylcholine transporter) and glial cell markers (anti-myelin basic protein). The primary antibodies used in the present study including working dilutions, RRID numbers, and suppliers are listed in Table1. The neurofilament antibody (AB5539, Merck-Millipore) is a polyclonal antibody and is raised against the bovine neurofilament heavy chain. By immunofluorescence, it has been demonstrated that this antibody visualizes myelinated and non-myelinated axons in humans (Gesslbauer etal. 2017; Blumer etal. 2020) and non-human tissue (Ghilardi etal. 2011; Tereshenko etal. 2021). The monoclonal mouse antibody against the nuclear neuronal protein (NeuN, MAB377, Merck-Millipore) labels cell nuclei in neurons and is the most widely used neuronal marker in neuroscience research and neuropathological assays (Gusel'nikova and Korzhevskiy 2015). The polyclonal goat antibody against choline acetyltransferase (ChAT, AB144P, Merck-Millipore) is raised against human placental ChAT. The antibody was validated in the human brain by pre-adsorption (Mesulam etal. 1992). By immunohistochemistry, it has been shown that this antibody visualizes cholinergic neurons in the human spinal cord (Gesslbauer etal. 2017) and brain (Lienbacher etal. 2019). The monoclonal anti-synaptophysin antibody clone SP15 (MAB329, Merck-Millipore) is validated for use in ELISA, Western blot, and immunofluorescence for the detection of the synaptic vesicle protein synaptophysin (manufacturer’s datasheet). The monoclonal antibody against MBP (MAB386) is raised against bovine myelin basic protein and was tested by Western blot in mouse brain (manufacturer’s datasheet). This antibody has been shown to visualize myelinated axons in the human spinal cord (Blumer etal. 2020) and non-human tissue (Nakanishi etal. 2005). The monoclonal antibody against calcitonin gene-related peptide (CGRP, 14959, Cell Signaling) visualizes CGRP in the central and peripheral nervous system. This antibody was validated in carcinoma patients exhibiting a correlation between perineural invasion and lymph node metastasis (Zhang etal. 2021). The VAChT antibody (139103 Synaptic system) is a polyclonal antibody and raised the against vesicular acetyl choline transporter, which is an integral protein of synaptic vesicles. This antibody was knockout-validated in mouse brains (Kljakic etal. 2021) and used to visualize cholinergic neurons in the human pancreas (Campbell-Thompson and Tang 2021). Table 1 List of primary and secondary antibodies used in the present study including working dilution, RRID number, and suppliers Primary antibodies Working dilution Catalog/RRID number Supplier General markers for axons/synapses/nerve cells Chicken anti-neurofilament 1:2000 AB5539/AB_177520 Merck/Millipore Mouse anti-synaptophysin 1:300 MAB329/AB_95786 Merck/Millipore Mouse anti-neuronal nuclear protein 1:500 1.1.1.1MAB377/AB_2298772 Merck/Millipore Marker for putative nociceptor fibers Rabbit anti-CGRP 1:1000 14959/AB_672598 Cell signaling technology Marker for myelin Rat anti-MBP 1:100 MAB386/AB_94975 Merck/Millipore Markers for cholinergic axons/synapses Goat anti-ChAT 1:100 AB144P/AB_2079751 Merck/Millipore Rabbit anti-VAChT 1:500 139103/AB_887864 Synaptic systems Fluorescent secondary antibodies Working dilution Catalog/RRID number Supplier Goat anti-chicken IgG AF568 1:500 A-11041/AB_2534098 Thermo Fisher Scientific Goat anti-chicken IgG AF647 1:500 A-21449/AB_2535886 Thermo Fisher Scientific Goat anti-mouse IgM AF488 1:500 A-21042/AB_141357 Thermo Fisher Scientific Goat anti-mouse IgM rhodamine 1:200 AP128R/AB11214322 Merck/Millipore Goat anti rabbit IgG AF488 1:500 A-11034/AB_2576217 Thermo Fisher Scientific Goat anti-rat IgG AF488 1:500 A-11006/AB_141373 Thermo Fisher Scientific Rabbit anti-chicken IgG rhodamine 1:200 SA1-9512/AB_1075135 Thermo Fisher Scientific Rabbit anti-goat IgG AF488 1:500 A11078/AB_141838 Thermo Fisher Scientific Biotinylated secondary antibodies Working dilution Catalog/RRID number Supplier Goat anti-rabbit IgG biotinylated Prediluted 21537/AB_916336 Merck/Millipore Rabbit anti-goat IgG biotinylated 1:500 65-6140/AB_2533969 Thermo Fisher Scientific Histochemistry and Cell Biology 1 3 Immunofluorescence We performed one single, four double, and three tripleimmunofluorescence stainings. Spinal cord sections were single-labeled with anti-neurofilament, double-labeled with anti-neurofilament plus (1) anti-ChAT, (2) anti-synaptophy- sin, (3) anti-CGRP, and (4) anti-NeuN, and triple-labeled using anti-neurofilament plus (5) anti-synaptophysin/anti- CGRP, (6) anti-synaptophysin/anti-VAChT, and (7) anti- CGRP/anti-MBP. In staining combination (4), we used bisbenzimide (Hoechst 33342, Thermo Fisher Scientific) to counterstain cell nuclei. Before immunolabeling, sections were air-dried for 30min. Then they were blocked for 1h with 10% normal rabbit serum (staining combination 1) or 10% normal goat serum (staining combinations 2–7) followed by incubation with the primary antibodies diluted in PBS containing 0.1% Triton X (PBS-T) for 48h at 4°C. After washing in PBS, sections were incubated with the secondary antibodies that were conjugated with different fluorochromes including Alexa Fluor (AF) 488, AF 568, and AF 647. The incubation time for the secondary antibodies was 2h at 37°C. Thereafter, sections were rinsed again in PBS and coverslipped with fluorescence mounting medium from Dako (DakoCytomation, Santa Clara, CA, USA). The fluorochrome conjugated secondary antibodies including working dilutions, RRID numbers, and supplier are listed in Table1. Horseradish peroxidase immunohistochemistry HRP immunohistochemistry was performed in ChAT and CGRP staining. Sections were pretreated with 1% H2O2 to suppress the endogenous peroxidase activity following incubation with 10% normal rabbit serum (ChAT staining) or 10% goat serum in PBS-T (CGRP staining). Thereafter, sections were incubated with the primary antibodies as described above. After washing in PBS, sections were incubated for 2h at 37°C with the biotinylated secondary antibodies rabbit anti-goat for the ChAT staining or goat antirabbit for the CGRP staining. After a further washing step, sections were incubated with avidin-neutravidin conjugated with HRP for 2h at 37°C. The antigenic site was visualized using 0.01% diaminobenzidine (DAB) containing 0.06% H202 in 0.1 PBS for 5 to 10min. Sections were coverslipped with aqueous mounting medium (Dako Cytomation). The biotinylated secondary antibodies including working dilutions, RRID numbers, and suppliers are listed in Table1. As a positive control, sections from the organ donor spinal cord were included in every staining combination and staining run. This approach allowed us to verify the validity of the staining process and exclude misinterpretation of results in the body donor spinal cord. Omission of the primary antibodies and using the secondary antibodies alone resulted in a complete lack of immunostaining. This confirmed that the secondary antibodies bind specifically to the primary antibodies. Microscopic imaging protocol Light microscopy Hematoxylin–eosin staining sections were analyzed with a slide scanner microscope (Olympus VS120, Olympus Europa SE & Co. KG, Hamburg, Germany). Sections labeled by the HRP immunohistochemistry technique were examined with a light microscope (Axioscope, Zeiss, Jena, Germany) and photo-documented using a digital camera (Nikon Eclipse 600 camera, Nikon, Tokyo, Japan) connected to the microscope. Confocal laser scanning microscopy Fluorescently labeled sections were analyzed with a confocal laser scanning microscope (CLSM Olympus FV3000). Images were captured with dry lenses of 4x and 20x magnification and oil lenses of 60x magnification. A series of virtual CLSM sections of 1-µm thickness were cut through the structures of interest. Each section was photo-documented with a 1024 × 1024 pixel resolution and 3D projections were rendered using ImageJ software (NIH, Bethesda, MD, USA). Single-colored images (Fig.2) were generated using a laser with excitation wavelength 633. Double-colored images (Figs.3, 4, 5, 6, 7, 8, 9) were generated using lasers with excitation wavelengths of 488 and 568nm, and triplecolored images (Figs.10, 11, 12) using additionally a laser with excitation wavelengths of 405 or 640nm. In certain cases, bright-field images were recorded in the CLSM to correlate immunolabeled structures to morphological structures (Fig.5a’’, b’’, c’’, d’’). Scanning strategy In all staining combinations and all postmortem groups, fluorescently labeled sections were analyzed and photodocumented immediately (within 1 day) after the staining experiments were completed. For every staining combination, individual CLSM settings were manually adjusted and care was taken to avoid oversaturation or undersaturation of images. Once found, settings parameters were kept constant for image acquisition of all samples in each staining combination and postmortem interval. This scanning strategy allowed us to determine changes in the signal intensity at different postmortem intervals. Histochemistry and Cell Biology 1 3 Histological quantification Axon counts To evaluate the accuracy of our immunofluorescence, we separately counted neurofilament positive axons and neurofilament/ChAT-positive axons in the anterior roots of the human spinal cord (N = 3). This was done in the spinal cords at 24 and 48h postmortem intervals. Whole cross sections of the anterior roots were scanned with a slide scanner [Axio Observer Z.1 microscope (Carl Zeiss Meditec AG, Jena, Germany)] followed by automated counts of axons using StrataQuest version 5.1.249 and Tissue Quest version 4.0.1.0128 (TissueGnostic Vienna, Austria). Quantification ofthestaining intensity The intensity of the antibody signals was quantified in human spinal cords with 2-h (N = 1), 24-h (N = 3), 48-h (N = 3), and 72-h (N = 3) postmortem intervals. The antibodies studied in each postmortem interval were anti-neu- rofilament, anti-ChAT, anti-CGRP, and anti-synaptophysin. For the quantification of the staining intensity, images of the specimens were captured at 20x magnification using identical CLSM settings. In each image, 25 randomly selected squares of 6 × 6μm were used to measure the intensity of the antibodies signal (i.e., optical density). In all cases, for background correction, ten optical density values of the same area were taken. Then, the background value was subtracted, and the values of the antibody optical density were normalized with respect to the 2-h postmortem spinal cord. The antibody staining intensity was set at 100% in the 2-h postmortem spinal cord and values for spinal cords with 24-, 48-, and 72-h postmortem intervals were calculated as the percentage of the 2-h postmortem control value. Statistical analyses The axon quantification data of double-positive axons (ChAT and neurofilament) and single-positive neurofilament axons were statistically compared using Student’s ttest at a level of significance of P < 0.05. The data of staining intensity (anti-neurofilament, anti-ChAT, anti-CGRP, and antisynaptophysin) of the different postmortem intervals (2, 24, 48, and 72h) were statistically compared using the one-way- ANOVA test followed by post hoc multiple comparisons (Holm–Sidak method) at a significance level of P < 0.05. All values were expressed as the mean ± standard error of the mean (SEM). Statistical analysis was carried out in Sigma Plot, version 11 (Systat Software, San José, CA, USA). Results Histology The histology of the spinal cord from body donors was analyzed using hematoxylin–eosin staining. The spinal cord at 24-h postmortem interval exhibited strong staining and the tissue architecture appeared normal. The two parts of the spinal cord, the inner H-shaped gray matter, and the outer white matter were clearly identifiable (Fig.1a). In line with a previous study, we observed that the anterior horn of the gray matter has a wide lateral expansion at the cervical level of the spinal cord (Kameyama etal. 1996). Forty-eight hours after death, small gaps were observed readily in the white and gray matter of the spinal cord (Fig.1b) and they increased in size and number 72h postmortem, indicating advanced autolysis of the tissue (Fig.1c). Moreover, 72h postmortem the staining intensity decreased and there was no sharp boundary between the spinal cord’s gray and white matter. Lipofuscin autofluorescence Lipofuscin is a lipid-containing degradation product and increases with age in neuronal and non-neuronal tissue (Brody 1960; Shiers etal. 2021). In the human brain, lipofuscin deposits were observed intraas well as extracellularly (Waldvogel etal. 2006). Although lipofuscin is virtually present in every type of neuron, it is most abundant in neurons initiating movements including motoneurons of the spinal cord (Gray and Woulfe 2005). Typically, lipofuscin exhibits strong red and green autofluorescence and can be recognized in a fluorescence microscope when using appropriate filters (Gray and Woulfe 2005). Concordant with this, we observed high lipofuscin autofluorescence in the motoneurons of the anterior horn of the organ donor and body donor spinal cords. Due to the older age, the lipofuscin accumulation was higher in body donors (mean age 81.2 ± 13.08years) than in the 39-year-old organ donor. The inset in Fig.2a highlights the anterior segment of the spinal cord, which is shown in immunofluorescent staining in Fig.2b’–b’’’. Figure2c–c’’ visualizes lipofuscin inside motoneuron cells bodies and processes in the neuropil. In motoneurons, the lipofuscin accumulation was graded and while few motoneuron cell bodies lacked lipofuscin, most cell bodies were filled to various degrees with this age-related cellular inclusion (Fig.2c–c’’). Because of the lipofuscin autofluorescence, care is necessary when interpreting immunofluorescence staining in the spinal cord and, at appropriate places, we referred to this point in the manuscript. Histochemistry and Cell Biology 1 3 Double immunofluorescence andperoxidase immunohistochemistry We performed four double-labeling immunofluorescence assays in the spinal cord of the organ donor and body donors at different postmortem intervals. Double immunofluorescence included labeling with anti-neurofilament plus (1) anti-ChAT, (2) anti-synaptophysin, (3) anti-CGRP, and (4) anti-NeuN. Alternatively, HRP immunohistochemistry for anti-ChAT and anti-CGRP was performed. Anti-ChAT is a marker for cholinergic neurons, anti-synaptophysin is a marker for synapses, anti-CGRP is a marker for putative nociceptor fibers, and anti-NeuN is a marker for the nuclei of neuronal cells. In our immunolabeling assays, some antibodies exhibited stronger signal intensity than others and we therefore adapted the CLSM settings for every individual staining combination. As a result of this approach, the red and green lipofuscin autofluorescence is visible in some figures (Figs.2b’–b’’’, c–c’’, 3b–b’’, 6a–a’’, b–b’’, 9b–b’’) whereas in other figures only red autofluorescence (Figs.7c, c’’, e, e’’, 8a, 10b–b’’, c–c”) or almost no autofluorescence is visible (Figs.10d–d”, 11e–e”, 11a, 12a–a’’, b–b’’). A supplementary figure (Supplementary Fig.1) is provided showing autofluorescence signal dependent on CLSM settings. Following anti-neurofilament/anti-ChAT incubation, neurofilament signals were observed in the gray and white Fig. 1 Hematoxylin–eosin staining of human spinal cords after different postmortem intervals. a Cross section of the spinal cord at 24h postmortem. The structure of the spinal cord appears normal without observable histological changes. Anterior roots (ar), posterior roots (pr). b, c Half of the spinal cord after 48h (b) and 72h (c) postmortem interval. At time point 48h, structural defects including small gaps (inset in b) are present in the spinal cord whereas at 72h, the gray and white matter exhibit large gaps, indicating advanced autolysis of the tissue (c). Scale bar 1000μm (a–c) Histochemistry and Cell Biology 1 3 matter of the spinal cord as well as in the anterior and posterior roots. ChAT signals were present in the anterior but not in the posterior roots of the spinal cord. The inset in Fig.3a highlights the segment of a 24-h body donor spinal cord, which is immunolabeled in Fig.3b–b’’. Images of the posterior root (Fig.3c–c’’) and anterior root (Fig.3d–d’’) are provided at higher resolution; 24-h postmortem, neurofilament, and ChAT signals were strong and almost equal to those observed in the spinal cord of the organ donor (Figs.4a–a’’, b–b’’, c–c’’, 5a–a’’, b–b’’). We combined fluorescence and bright-field images to match fluorescently labeled structures with anatomical structures. By this technique it was possible to visualize the myelin sheath surrounding the fluorescently labeled axons of the anterior root (Fig.5a’’, b’’). Fig. 2 Human spinal cord labeled with anti-neurofilament (NF) after 24 h postmortem interval showing lipofuscin autofluorescence. a Scheme with an inset illustrating the part of the spinal cord shown in figure b–b’’’. Posterior root (pr), anterior root (ar). b–b’’’ Spinal cord in neurofilament staining (b), red autofluorescence (b’), green autofluorescence (b’’), and overlay (b’’’). Thethick dashed line demarcates the gray and white matter and thethin dashed lines the surface of the spinal cord and the anterior root. b Grey and white matter and the anterior root (ar) express neurofilament. b’ Motoneurons of the anterior horn exhibit red and b’’ green autofluorescence due to lipofuscin deposits. b’’’ The overlay of b–b’’ shows that the mixture of red and green autofluorescence results in yellow color. c–c’’ High magnification of motoneurons of anterior horn demarcated with a dashed line showing neurofilament signal and red lipofuscin autofluorescence (c), neurofilament and green lipofuscin autofluorescence (c’), and the overlay of c–c’ (c’’). The amount of lipofuscin varies and two cell bodies are fully covered with lipofuscin, one cell partly, and one cell (arrow) is free of lipofuscin. Lipofuscin granules exhibiting red and green autofluorescence are also visible outside the cell bodies in the neuropil.Scale bars 500µm in b’’’ for b–b’’’ and 50µm in c’’ for c–c’’ Histochemistry and Cell Biology 1 3 After 48- and 72-h postmortem intervals, the neurofilament and ChAT signals decreased. The signal reduction was moderate in the neurofilament but strong in the ChAT staining as demonstrated for the anterior roots (Fig.4d–d’’, e–e’’, low resolution; Fig.5c–c’’ and d–d’’, high resolution). Forty-eight hours after death, few axons completely lacked Fig. 3 Double labeling with anti-neurofilament (NF) and anti-ChAT after 24 h postmortem interval. a Scheme with an inset illustrating the part of the spinal cord shown in figure b–b’’. Posterior roots (pr), anterior roots (ar). b The spinal cord in neurofilament staining. The gray and white matter and the anterior (ar) and posterior (pr) roots express neurofilament. Thedashed line demarcates the gray and white matter. Motoneurons of the anterior horn exhibit red autofluorescence due to lipofuscin deposits (arrow). b’ The spinal cord in ChAT staining. Fascicles of the anterior root exhibit ChAT immunoreactivity whereas fascicles of the posterior root lack ChAT immunoreactivity. Thegreen pial contour covering the surface of the posterior root represents an artificial signal. Motoneurons of the anterior horn exhibit green autofluorescence due to lipofuscin (arrow). b’’ Overlay of b and b’, shows that the nerve fascicles of the anterior root are positive for neurofilament and ChAT. c–c’’ The fascicles of the posterior root (inset in the upper part of c’’). Axons of the posterior root are neurofilament positive (c) but lack ChAT (c’). In the overlay (c’’), neurofilament and ChAT staining is merged. d–d’’ Fascicles of the anterior root (inset in the lower part of b’’). Axons of the anterior root express neurofilament (d) and ChAT (d’). In the overlay d’’, the mix of the red neurofilament and green ChAT signal results in yellow color.Scale bars: 1000µm in b’’ for b–b’’; 100µm in d’’ for c–c’’ and d–d’’ Histochemistry and Cell Biology 1 3 neurofilament/ChAT signals, but this was more frequent 72-h postmortem (arrows in Fig.5c–c’’, d–d’’). Additionally, by 72h, most axons revealed less clear delineation coupled with incomplete labeling (Fig.5d–d’’). We quantified the staining intensity in the 2-h postmortem spinal cord of the organ donor (N = 1) and in the spinal cords of the body donors at every postmortem interval (N = 3 for each postmortem interval). Measurements are percentages relative to the 2-h spinal cord value, which was the reference value and set at 100% (Fig.4f, left bar; 4g left bar). At time point 24h, the neurofilament staining intensity was 90% of the 2-h reference value (Fig.4f, second bar). At the time point of 48h, the neurofilament signal was at about 70% (Fig.4f, third bar) and at 72h at around 45% of the reference value (Fig.4f, fourth bar). Relative to the reference value, the ChAT signal was 93% at time point 24h (Fig.4g, second bar), 40% at time point 48h (Fig.4g, third bar), and less than 20% at 72h (Fig.4g, fourth bar). We evaluated the precision of the neurofilament/ChAT immunolabeling by counting separately neurofilament and neurofilament/ChAT-positive axons in the anterior root of the 24- and 48-h spinal cord (N = 3 for each point of time). Because the anterior roots of the human spinal cord carry exclusively cholinergic (motor) axons, all neurofilamentpositive axons in the anterior root should express ChAT as well. Automated quantification revealed that at time point 24h, 99.5% of the axons in the anterior root expressed ChAT (Fig.4h, left bars) and this was confirmed by high-resolution images, as there was a complete match of neurofilament/ ChAT signals (Fig.5b–b’’). The small gap to 100% could be explained that the ChAT signal varies along the axons (Zimmermann etal. 2013) and it is possible that axons with weaker ChAT signals were missed by the analysis software. At time point 48h, significantly fewer ChAT-positive axons (90.3%) were counted (Fig.4h, right bars). This finding proves that immunolabeling was very precise 24h postmortem but no longer 48h postmortem. Although the ChAT signals were strong and almost equal in the motor axons 2-h and 24-h postmortem, ChAT immunoreactivity in motoneuron cell bodies was observed 2h but not 24h postmortem (Fig.6a–a’’, b–b’’). Interestingly, the ChAT intensity was lower in motoneuron cell bodies than in motoneuron axons. It is important to note that the ChAT signal in motoneuron cell bodies was only visible in the green fluorescence but not in the red fluorescence channel of the CLSM (Fig.6a–a’’), thus excluding being lipofuscin autofluorescence, which would be detectable in both fluorescence channels. We therefore conclude that the ChAT immunoreactivity in motoneuron cell bodies 2h postmortem was specific. Different from that pattern, motoneuron cell bodies in the 24-h postmortem spinal cord exhibited signals in the green fluorescence and red fluorescence channel, indicating lipofuscin autofluorescence instead of a specific ChAT signal (Fig.6b–b’’). Interestingly, cholinergic synapses, which were observed on motoneurons, exhibited bright ChAT signals 2 and 24h postmortem (Fig.6a’–a’’, b–b’’). The lack of ChAT signals in motoneuron cell bodies 24h postmortem might be explained by the fact that the ChAT enzyme became dissociated or alternatively was at a level too low for detection by immunofluorescence. We therefore performed HRP immunohistochemistry because there are examples in the literature that this technique is more sensitive than immunofluorescence (Molne etal. 2005). By using this approach, we observed robust ChAT immunoreactivity in the motoneurons of the 2-h donor spinal cord (Fig.6c, e), whereas in the 24-h body donor spinal cord, weak signals were found in some but not all motoneurons (Fig.6d, f). Analog to immunofluorescence, cholinergic synapses exhibited a high signal intensity in immunoperoxidase staining (arrows in Fig.6c, d, f). We next performed labeling with anti-neurofilament and anti-synaptophysin. Synaptophysin is a protein present in synaptic contacts (De Camilli etal. 1988) and synaptophysin was observed throughout the spinal cord’s gray matter as demonstrated in a panoramic image of the body donor spinal cord 24h postmortem (Fig.7a). Synaptophysin was also found along cell processes (dendrites) extending from the gray into the white matter (arrows in Fig.7a). We observed synaptophysin signals at the periphery of the spinal cord and in the posterior part of the spinal cord along the midline (Figs.7a, 10a). Microscopic analyses showed that the fluorescence signal corresponded with plaques in the white matter. To evaluate the quality of the synaptophysin labeling along postmortem intervals, we examined synaptic contacts on spinal cord motoneurons. We observed no differences between 2 and 24h postmortem, and synapses expressing high levels of synaptophysin covered the surface of the motoneuron cell bodies (Fig.7b–b’’, c–c’’). Forty-eight hours after death, the signal brightness was reduced but the synaptic density on motoneuron cell bodies appeared unchanged (Fig.7d–d’’). At time point 72h, the signal intensity further decreased and the synaptic density was reduced (Fig.7e–e’’). Morphologically, large tissue-free gaps surrounded the motoneurons 72h postmortem (Fig.7e–e’’). Measurements of the staining intensity demonstrated that the synaptophysin signal was almost equal in the 2-h postmortem spinal cord of the organ donor and in the body donor spinal cord 24h postmortem (Fig.7f). At time point 48h, the signal intensity of synaptophysin was reduced to about 60% of the 2-h value (Fig.7f) and at time point 72h to approximately 25% of the 2-h value (Fig.7f). After labeling the spinal cords of the organ donor and body donors with anti-neurofilament/anti-CGRP, we observed CGRP expression in the superficial layer of the posterior horn with decreasing CGRP signals in deeper Histochemistry and Cell Biology 1 3 signal reduction observed here was antibody-specific and strongest in the anti-ChAT antibody. This signal reduction had consequences when performing quantitative analyses. Specifically, 48h postmortem only 90 out of 100% of cholinergic axons in the anterior root were identified, indicating that in the missing axons, ChAT signals were at a level too weak for being detected by the analysis software. These findings along different postmortem intervals indicate that qualitative immunohistochemical information can be obtained from body donor neuronal tissue over a Fig. 8 Double labeling with anti-neurofilament (NF) and anti-CGRP. a A segment of the human spinal cord at 24-h postmortem interval. Intense CGRP immunoreactivity is seen in the superficial layer of the posterior horn. Thedashed line demarcates the gray and white matter of the spinal cord. Anterior roots (ar), posterior roots (pr). Motoneuron cell bodies exhibiting lipofuscin autofluorescence are indicated by an arrow. b–e The CGRP-immunoreactivity in the dorsal horn of the organ donor (b) and body donors (c–e) at different postmortem intervals. At time points 2h (b) and 24h (c), a high level of CGRP is seen in the superficial layer of the dorsal horn. There is a downward trend in the staining intensity at 48h (d) and at 72h (e). Several tissue gaps are present 72h postmortem (e). Scale bars: 1000µm in a, 100µm in b, and 100µm in e for c, d, and e. f Bar chart showing the reduction of the CGRP signal over the postmortem interval. At time point 24h, the CGRP intensity is 85% of the 2-h value (f, second bar). At time point 48h, the CGRP signal intensity is 55% (f third bar) and at 72h at about 30% (f, third bar) of the initial value (f, first bar).Black dots (●) indicate significant differences with time point 2h,asterisks (*) indicate significant differences with time point 24 h, andplus signs (+) mark significant differences with time point 48h. (one-way ANOVA test followed by Holm–Sidak post hoc method, P < 0.05) Histochemistry and Cell Biology 1 3 period longer than 24h after death but there are limitations regarding quantitative analyses. Based on our observations, a postmortem interval of 72h is too long for proper immunohistochemistry in neuronal tissue from body donors. At this time point, spinal cords exhibited substantial morphological changes due to autolysis and with the exception of the neurofilament antibody, immunohistochemical signals in the other antibodies (anti-ChAT, anti-synaptophysin, and anti-CGRP) were very low i.e., between 20 and 40% of the initial value. These postmortem changes are consistent with findings in the brain, where the synaptophysin signal significantly decreased 72h after death (Liu and Brun 1995), and do not support data in autonomic nerve fibers of the heart where the signal drop was observed not until the 7th day postmortem (Chow etal. 1998). The low tissue quality coupled with strong immunohistochemical signal loss questions the scientific value of information obtained from human neuronal tissue 72h after death. The antibody-specific signal reduction observed in the present study is consistent with two older publications evaluating the postmortem effects on the immunohistochemistry of autonomic nerve fibers in humans (Gu etal. 1985; Chow Fig. 9 Double fluorescent labeling with anti-neurofilament (NF) and anti-CGRP and HRP immunohistochemistry with anti-CGRP. a–a’’, b–b’’ Immunofluorescence of motoneurons stained with anti-neu- rofilament and anti-CGRP 2h (a–a’’) and 24h postmortem (b–b’’). Dashed lines demarcate motoneuron cell bodies. At time point 2 h (a’), motoneurons exhibit weak CGRP signals but CGRP signals are not visible 24h postmortem (b’). Lipofuscin (LP) exhibiting red and green autofluorescence fills a part of the cell body (b, b’). Overlays of the neurofilament (a, b) and CGRP (a’, b’) staining are presented in a’’, b’’. The overlay shows lipofuscin autofluorescence in orange color due to the mix of the strong red and green autofluorescence signal (b”).Scale bar: 20µm in b’’ for a–a’’ and b–b’’. c, d HRP immunohistochemistry with anti-CGRP showing motoneuron cell bodies 2 h (c) and 24 h postmortem (d). Clod-like CGRP signals appear inbrown color and are visible at both postmortem intervals in cell bodies. In c, a cell body without a CGRP signal (arrow) is present. Lipofuscin (LP).Scale bar: 20µm in d for c and d Histochemistry and Cell Biology 1 3 Fig. 10 Triple labeling with anti-neurofilament (NF), anti-NeuN, and Hoechst 33342. a Schematic drawing with an inset illustrating the part of the spinal cord shown in figures b–b’’, and d–d’’. Posterior roots (pr), anterior roots (ar). b–b’’, d–d’’ Overview images of the anterior horn 2h (b–b’’) and 24h postmortem (d–d’’) are shown in neurofilament and NeuN staining (b, d), neurofilament and Hoechst staining (b’, d’), and overlays (b’’, d’’). The gray and white matter are separated by adashed line. Motoneuron nuclei of the anterior horn express NeuN immunoreactivity (arrowheads in b, d). The inset in d shows small nuclei positive for NeuN (arrowheads). c–c’’, e–e’’ High magnification images of motoneurons cell bodies (surrounded by adashed line) 2h (d–d’’) and 24h postmortem (e–e’’) in neurofilament and NeuN staining (c, e), neurofilament and Hoechst staining (c’, e’) and overlay (c’’, e’’). The motoneuron nucleus expresses NeuN (c, e) and is visualized by Hoechst stain in c’ and e’. The overlays of c and c’ and e and e’ show co-localization of NeuN and the Hoechst signal. Small nuclei expressing NeuN (arrowheads in c, e) and Hoechst signals (c’, e’) are also visible.Scale bar: 200µm in b’’ for b–b’’; 15µm in c’’ for c–c’’; 200µm in d’’ for d–d’’; 15µm in e’ for e–e’’ Histochemistry and Cell Biology 1 3 etal. 1998) and a recent study evaluating this effect in the brain (Waldvogel etal. 2006; Blair etal. 2016). After death, tissue proteins degrade and this reduces the binding capacity of antibodies and consequently the signal strength (Ramos- Vara and Miller 2014). Because the degradation of tissue proteins is temperature dependent, it is important to refrigerate body donors after arrival at body donor facilities. The stability of different tissue proteins varies in postmortem tissue (Waldvogel etal. 2006) and there are indications that structural proteins like neurofilament (Karlsson etal. 1991) and cell membrane proteins like synaptophysin (Wiedenmann and Franke 1985) are better preserved than soluble enzymes (ChAT) (Bruce and Hersh 1987). This was confirmed in the present study because the signal loss was less dramatic in neurofilament and synaptophysin staining compared to ChAT staining. CGRP a neuromodulator protein is similar well preserved as synaptophysin as shown here. An additional discovery of the present study was that the signal intensity of the ChAT antibody varied in different compartments of the motoneuron. Specifically, in the 24-h spinal cord high levels of ChAT were present in the axons of the anterior root and in the synapses of the motoneuron cell bodies whereas low or no ChAT signal was inside the motoneuron cell bodies. This spatial variation Fig. 11 Triple labeling of a body donor spinal cord 24h postmortem with anti-neurofil- ament (NF), anti-synaptophysin (syn), and anti-CGRP. a Cross section of a body donor spinal cord at 24h postmortem interval. The whole gray matter exhibits synaptophysin immunoreactivity whereas CGRP is expressed in the superficial layer of the posterior horn. Anterior roots (ar), posterior roots (pr). b–b’’ High-resolution images of the posterior horn (square box in a) in neurofilament/synaptophysin staining (b) neurofilament/CGRP staining (b’) and overlay b’’. The colocalization of synaptophysin (red) and CGRP (green) results in yellow color (b’’).Scale bars: 1000µm in a, 100µm in b’’ for b–b’’ Histochemistry and Cell Biology 1 3 of the staining intensity suggests that the ChAT enzyme disintegrates postmortem with variable speed in different motoneuron compartments and based on the low antibody signal, this process is quicker in cell bodies than in axons and synapses. Taken together, information on the stability of different neuronal proteins and variations of the protein stability in neuronal compartments is crucial and must be considered when evaluating immunohistochemistry in human postmortem tissue. Fig. 12 Triple labeling of a body donor spinal cord 24 h postmortem. (a–a’’, b–b’’) Labeling with anti-neurofilament (NF), antisynaptophysin (Syn), and anti-VAChT. Cross section of the anterior horn (a–a’’) showing motoneurons (arrows in a) covered with synaptophysin (a) and VAChT (a’) positive synapses. The dashed line demarcates the border between the white and gray matter. The overlay (a’’) shows the mix of neurofilament, synaptophysin, and VAChT staining. (b–b’’) High-resolution image of a motoneuron cell body (*) covered with synapses expressing synaptophysin (b) and VAChT (b’). The overlay (b’’) shows colocalization of synaptophysin/VAChT in some synapses which appear in yellow color. (c–c’’) Labeling of the posterior root with anti-neurofilament, anti-MBP, and anti-CGRP. Most axons are covered with a myelin sheath (c) and only a few axons (arrows in c) are without myelin. Some thin axons express CGRP (c’) and are with myelin (arrowheads in c’’) or without a myelin sheath (arrows in c’’). Scale bars: 100µm in a’’ for a–a’’, 25µm in b’’ for b–b’’, 10µm in c’’ for c–c’’ Histochemistry and Cell Biology 1 3 Conclusions Here, we have demonstrated that multi-color immunofluorescence coupled with HRP immunohistochemistry allows detailed qualitative and quantitative analyses in the spinal cord from body donors 24h postmortem and at least qualitative analyses are possible until 48h postmortem. These data underline that neuronal tissue from body donors can be an alternative source for immunohistochemical analyses in basic and clinical research. Moreover, qualitative and quantitative immunohistochemical information obtained from normal neuronal tissue of body donor can be compared with immunohistochemical information from cases with neuronal disorders. Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s00418- 022- 02154-5. Acknowledgements The authors express their thanks to the people who donated their bodies to the Division of Anatomy of the Medical University of Vienna. Further, the authors wish to thank Sylvia Gerges, Alina Ehrntraut, and Sandra Trojanek for their helpful technical assistance. Author contributions Study design: LR, WJW, and RB. Tissue harvesting: LR and AH. Immunofluorescence analyses: GC-R and RB. Histology and quantitative analyses: ASM, GC-R, and UM. Manuscript preparation: GC-R and RB. All authors analyzed data and made critical revisions and approved the manuscript. Funding Open access funding provided by Medical University of Vienna. Declarations Conflict of interest All the authors declare that there are no conflicts of interest. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. 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