Laser capture microdissection protocol for xylem tissues of woody plants

pls-07-01965 Decembe 27, 2016 Time: 12:11 # 1 PROTOCOLS published: 04 Janua y 2017 doi: 10.3389/ pls.2016.01965 Edi ed by: Roge Deal, Emo y Uni e si y, USA Re iewed by: Michael J. Scanlon, Co nell Uni e si y, USA Pascal Gamas, Cen e Na ional de la Reche che Scien i ique, F ance *Co espondence: Ku Fage s ed [email p o ec ed] To e Nii ylä [email p o ec ed] †P esen add ess: Anna Kä könen, Na u al Resou ces Ins i u e Finland (Luke), G een Technology, La oka anonkaa i 7, Helsinki, Finland ‡These au ho s ha e con ibu ed equally o his wo k. Special y sec ion: This a icle was submi ed o Technical Ad ances in Plan Science, a sec ion o he jou nal F on ie s in Plan Science Recei ed: 24 Oc obe 2016 Accep ed: 12 Decembe 2016 Published: 04 Janua y 2017 Ci a ion: Blokhina O, Vale io C, Sokołowska K, Zhao L, Kä könen A, Nii ylä T and Fage s ed K (2017) Lase Cap u e Mic odissec ion P o ocol o Xylem Tissues o Woody Plan s. F on . Plan Sci. 7:1965. doi: 10.3389/ pls.2016.01965 Lase Cap u e Mic odissec ion P o ocol o Xylem Tissues o Woody Plan s Olga Blokhina1‡, Conce a Vale io2,3‡, Ka a zyna Sokołowska3,4, Lei Zhao1, Anna Kä könen5†, To e Nii ylä3*and Ku Fage s ed 1* 1Viikki Plan Science Cen e, Depa men o Biosciences, Uni e si y o Helsinki, Helsinki, Finland, 2Plan S ess Signaling, Ins i u o Gulbenkian de Ciência, Oei as, Po ugal, 3Umeå Plan Science Cen e, Depa men o Fo es Gene ics and Plan Physiology, Swedish Uni e si y o Ag icul u al Sciences, Umeå, Sweden, 4Depa men o Plan De elopmen al Biology, Ins i u e o Expe imen al Biology, Uni e si y o W ocław, W ocław, Poland, 5Viikki Plan Science Cen e, Depa men o Ag icul u al Sciences, Uni e si y o Helsinki, Helsinki, Finland Lase cap u e mic odissec ion (LCM) enables p ecise dissec ion and collec ion o indi idual cell ypes om complex issues. When applied o plan cells, and especially o woody issues, LCM equi es ex ensi e op imiza ion o o e come such ac o s as igid cell walls, la ge cen al acuoles, in e cellula spaces, and echnical issues wi h hickness and la ness o he sec ions. He e we p esen an op imized p o ocol o he lase -assis ed mic odissec ion o de eloping xylem om ma u e ees: a gymnospe m (No way sp uce, Picea abies) and an angiospe m (aspen, Populus emula) ee. Di e en cell ypes o sp uce and aspen wood (i.e., ay cells, achea y elemen s, and ibe s) we e success ully mic odissec ed om angen ial, c oss and adial c yosec ions o he cu en yea ’s g ow h ing. Two app oaches we e applied o achie e sa is ac o y la ness and ana omical in eg i y o he sp uce and aspen specimens. The commonly used memb ane slides we e ine ec i e as a moun ing su ace o he wood c yosec ions. Ins ead, in he p esen p o ocol we use glass slides, and in oduce a glass slide sandwich assembly o he p epa a ion o aspen sec ions. To asce ain ha no only he ana omical in eg i y o he plan issue, bu also he molecula ea u es we e no comp omised du ing he whole LCM p ocedu e, good quali y o al RNA could be ex ac ed om he mic odissec ed cells. This showed he e iciency o he p o ocol and es ablished ha ou me hodology can be in eg a ed in ansc ip ome analyses o elucida e cell-speci ic molecula e en s egula ing wood o ma ion in ees. Keywo ds: c yosec ion, lase cap u e mic odissec ion, ay cells, RNA in eg i y, acheids, xylem ibe s INTRODUCTION Lase cap u e mic odissec ion (LCM) was o iginally de eloped o assis semi-au oma ic collec ion o indi idual cells and issues om complex biological specimens o subsequen genomic and ansc ip omic analyses (Ho ne e e al., 2007). The me hod allows p ecise analysis o a ge cells, and is con ac - and con amina ion- ee (Gau am and Sa ka , 2015). LCM is a mic oscope- based echnique and in ol es se e al c ucial s eps: sample p epa a ion (sample embedding and sec ioning, o c yosec ioning), isualiza ion o in ac cells o issues o be isola ed, ou lining and lase -cu ing o he selec ed a eas and, inally, collec ion o dissec ed cells ei he by g a i y in o a ube, o by ca apul ing he a ge cells agains g a i y in o an adhesi e cap o a collec ion ube (Ho ne e e al., 2007). F on ie s in Plan Science | www. on ie sin.o g 1Janua y 2017 | Volume 7 | A icle 1965 pls-07-01965 Decembe 27, 2016 Time: 12:11 # 2 Blokhina e al. Lase Cap u e Mic odissec ion P o ocol o Xylem De elopmen o new echniques o high- h oughpu da a acquisi ion, concu en de elopmen o bioin o ma ic analyses and inc easing a ailabili y o sequenced genomes ha e made LCM a me hod o choice o elucida ing he cell ype- speci ic molecula ea u es. Downs eam applica ions can co e i ually e e y aspec o cell biology. Abo e all, gene exp ession analyses a e, nowadays, o en pe o med h ough genome-wide exp ession p o iling based on mic oa ay o nex gene a ion sequencing (NGS) echnologies (whole genome sequencing, a ge ed genome sequencing, RNA sequencing and ibosome p o iling). The combina ion o LCM wi h NGS echniques is a powe ul app oach o s udy cell- and issue-speci ic p ocesses (Abbo e al., 2010;Jyske e al., 2015). Mic odissec ion o plan issues and indi idual cells poses a challenge due o he p esence o a cell wall, a la ge cen al acuole in mos ully di e en ia ed cells and, in some issues, la ge in e cellula spaces (Nakazono e al., 2003). The LCM echnique, which was o iginally de eloped o animal issues, has equi ed subs an ial op imiza ion o plan s udies. Asano e al. (2002) we e he i s o apply LCM on plan issues in a s udy o phloem- speci ic gene exp ession in ice (O yza sa i a). Ex ensi e wo k was la e pe o med o es ablish p o ocols on plan issues, mainly on he baceous species and by using pa a in-embedded ma e ial (Ke k e al., 2003;Inada and Wilde mu h, 2005;Rajhi e al., 2011;Shiono e al., 2014). Fo example, p e- ixed pa a in sec ions o maize (Zea mays) oo s we e analyzed o di e en ial gene exp ession be ween epide mal cells and ascula issues using a mic oa ay (Nakazono e al., 2003). The same expe imen al app oach has been applied in s udies on ae enchyma o ma ion in he co ex o maize oo s (pa a in sec ions on memb ane slides) (Rajhi e al., 2011), and on sube iniza ion o oo s du ing o ma ion o a ba ie o adial oxygen loss in ice (Shiono e al., 2014). The indings o he Nakazono g oup on LCM o pa a in- embedded issues ha e been summa ized in a me hodological a icle by Takahashi e al. (2010). Non-pa a in embedding p ocedu es ha e been in oduced ecen ly o LCM o plan issues. An al e na i e s a egy o sample p epa a ion which gua an eed RNA quali y and mo phological in eg i y, eplaces pa a in wi h S eedman’s wax. This polyes e wax has a lowe mel ing poin han pa a in (38−40◦C s 60−65◦C), and can be washed ou by e hanol, an o e all milde sample ea men han he pa a in emo al by xylene. The S eedman’s wax-embedding has been pe o med, o example, in s udies on a buscula myco hiza and oo co ical cells o ba el medic (Medicago unca ula) (Gomez e al., 2009; Hogekamp e al., 2011). Good esul s in e ms o RNA quali y and RNA sequencing ha e been obse ed also a e incuba ion o plan ma e ial in polye hylene glycol (PEG)-e hanol mix u e and subsequen inclusion in PEG-modi ied S eedman’s wax and polye hylene naph ala e (PEN)-coa ed slides o LCM (Roux e al., 2014). This p o ocol was de eloped u he and 100% PEG was used as an embedding medium wi h sec ions moun ed on PEN slides (Ja dinaud e al., 2016). Much hicke longi udal oo sec ions (35 µm) o ba el medic ha e been p epa ed o LCM using embedding in issue- eezing medium and collec ed myco hizal ma e ial was used o gene exp ession p o iling (Gaude e al., 2012). LCM has also been used o s udy issue- and cell-speci ic p ocesses o non-xylem issues in woody plan s. Abbo and co-wo ke s success ully isola ed esin duc s and cambial issues om angen ial c yosec ions o 2-yea -old whi e sp uce (Picea glauca) plan le s. This allowed he cha ac e iza ion o RNA, de e mina ion o enzyme ac i i y and e penoid me aboli es (Abbo e al., 2010). S e eo mic oscope-assis ed dissec ion o c yosec ions ollowed by mic ogenomics has been used in a unc ional s udy o neighbo ing me is ema ic cells in he ascula cambium o Populus species (Goué e al., 2008, 2012). Jyske and co-wo ke s (Jyske e al., 2015) ha e applied he LCM echnology o assess he seasonal a ia ion in he phloem s uc u e and i s cons i uen s in ma u e No way sp uce (Picea abies) ees. LCM has been used also o in es iga e gene exp ession in phloem o No way sp uce (Nagy e al., 2014). In all hese s udies, he so phloem, cambial o co ex issues appea ed o be sui able o p epa a ion o samples h ough ei he c yosec ioning o pa a in- embedding o LCM. Xylem issue wi h i s highly ligni ied cell walls has p o en challenging o LCM applica ions. La isch e al. (2012) used LCM o sepa a e de eloping and ma u e wood om wigs o popla (Populus ×canescens), and subsequen ly analyzed he ma u e wood ansc ip ome using whole-genome mic oa ays (La isch e al., 2012). The au ho s a gued ha ay cells, as he main li ing cells in ma u e wood, con ibu ed mos o he isola ed RNA. Howe e , he me hod did no allow ay-cell-speci ic isola ion o RNA due o p oblems wi h RNA deg ada ion and yield. Recen ly, LCM was also used o p epa e ay cell-wall-en iched ma e ial o lignin composi ion analysis om he wood o Amu co k ee (Phellodend on amu ense) (Zheng e al., 2016). This analysis p o ided a cell speci ic lignin p o ile, bu no RNA was isola ed. The s udies o La isch e al. (2012) and Zheng e al. (2016) illus a e he po en ial o LCM me hod, while a he same ime highligh ing he need o a obus xylem-cell-speci ic LCM p o ocol which does no comp omise RNA quali y. He e we de eloped a p o ocol o LCM o de eloping xylem issues om 40-yea -old No way sp uce (Picea abies) and aspen (Populus emula) ees, ep esen a i es o gymnospe ms and angiospe ms. The wo k wi h he gymnospe m ee was done a he Viikki Plan Science Cen e in Finland, and wi h he angiospe m ee a he Umeå Plan Science Cen e in Sweden. The main goal was o achie e high quali y RNA om di e en xylem cell ypes ( ay pa enchymal cells, xylem ibe s and xylem achea y elemen s (i.e., acheids in sp uce) sui able o high- h oughpu analyses. Special emphasis was placed on he isola ion o ay pa enchymal cells, while a oiding collec ing ay acheids ypical o sp uce. Sligh ly di e en app oaches we e ound o be op imal o he p epa a ion and handling o sp uce and aspen samples p io o LCM. Du ing p epa a ion o he sec ions we es ed a ying sample hicknesses and o ien a ions. We used PEN-coa ed slides and plain glass slides, choosing he la e as he bes moun ing su ace o ix he c yosec ions. We es ablished di e en dehyd a ion p ocedu es o No way sp uce and aspen. Co ec handling o he samples p io o c yosec ioning was c ucial o he ana omical and RNA in eg i y. Se e al me hodological p oblems we e o e come du ing he me hod de elopmen . These included: di icul y in achie ing F on ie s in Plan Science | www. on ie sin.o g 2Janua y 2017 | Volume 7 | A icle 1965 pls-07-01965 Decembe 27, 2016 Time: 12:11 # 3 Blokhina e al. Lase Cap u e Mic odissec ion P o ocol o Xylem su icien la ness o he sec ions on slides, s a ic elec ici y, e y low RNA yield, and op imiza ion o lase powe o cu ing specimens in cell wall- ich a eas in compa ison wi h he cy osolic pa o he cells. These me hodological issues oge he wi h some use ul ips a e add essed in he ‘c i ical no es’ sec ion and in he oubleshoo ing able. P o ocol O e iew We de eloped p o ocols o he LCM o wood sec ions o ex ac ion o cell ype-speci ic o al RNA om de eloping xylem o No way sp uce and aspen. Disks o wood s ems o 40-yea - old sp uce and aspen ees we e collec ed du ing he ac i e seconda y g ow h and s o ed a −80◦C. Fo an easie handling o he samples du ing p epa a ion o he c yosec ions, he s em disks we e u he sawn in o smalle blocks. The blocks we e hen used o p epa e 30/40 µm hick angen ial (sp uce) o c oss and adial (aspen) sec ions using a c yomic o ome. The c yosec ions, moun ed on mic oscope glass slides, we e ixed wi h e hanol. Sp uce and aspen wood equi ed di e en p ocedu es o dehyd a ion and la ening o he sec ions. Sp uce angen ial sec ions we e la ened wi h a pipe e ip du ing he las dehyd a ion s ep in 100% e hanol. Aspen c oss and adial sec ions we e ixed in 75% e hanol and hen la ened using a mic oscope slide sandwich du ing dehyd a ion in a eeze- d ye chambe . In insic di e ences in wood ana omy be ween gymnospe ms and angiospe ms de e mined he sec ioning plane and, subsequen ly, he la ening me hod o he c yosec ions. Fo example, he p esence o la ge essels in aspen p e en ed he use o angen ial sec ions, because o he high p obabili y o sec ion b eakage along he essel. Indi idual cell ypes ( ay pa enchymal cells, acheids, and ibe s) we e isola ed by LCM using a PALM Mic o-Beam mic oscope a oom empe a u e, and collec ed in o adhesi e caps o collec ing ubes. Dissec ed ma e ial was snap- ozen in liquid ni ogen and s o ed a −80◦C un il RNA isola ion. Speci ic cell ypes collec ed o e se e al LCM sessions we e pooled oge he o RNA ex ac ion. Ad ances o he P o ocol – A comp ehensi e LCM p o ocol o he de eloping xylem o bo h angio- and gymnospe ms, applicable o angen ial, adial and c oss sec ions. – Less labo ious p o ocol, a oiding long embedding p ocedu es common in se e al published p o ocols. – Replaces memb ane-coa ed slides wi h glass mic oscope slides, and in oduces he easy assembly o a “glass sandwich” o keep ana omical in eg i y and la ness o he xylem sec ions. – Enables isola ion o eache y elemen s, ibe s and ay cells o RNA ex ac ion. – Enables isola ion o cell- ype speci ic, good quali y o al RNA o high- h oughpu analyses. STEPWISE PROCEDURES The me hod is di ided in o i e s ages om A o E: (A) issue sampling, handling and p epa a ion o he ozen specimens; (B) c yosec ioning; (C) ixa ion and dehyd a ion; (D) LCM; (E) RNA ex ac ion and quali y assessmen (Figu e 1). We adjus ed some o he s eps o he p o ocol aking in o accoun di e en ea u es o he wo woody ma e ials, in o de o es ablish he easies and as es echnical p ac ice. The goal was o ex ac good quali y RNA om he LCM-dissec ed cells, and hence, special ca e was aken o educe he isk o RNA deg ada ion du ing each s ep o he p o ocol. S age A. Tissue Sampling, Handling, and P epa a ion o he F ozen Specimens Time: 30 min Tempe a u e: oom empe a u e, o in liquid ni ogen No way Sp uce A 40-yea -old sp uce ee was elled du ing ac i e seconda y g ow h. Disks o he unk we e sawn and immedia ely ozen in d y ice, and hen s o ed in plas ic bags a −80◦C. In o de o a oid hawing and o acili a e he subsequen p epa a ion o c yosec ions, small wood blocks we e cu om he ozen s em disks wi h a jigsaw. Fo con enience, he s em disks we e i s sawn in hal es. Fu he sawing was conduc ed in wo pa s: i s , a long s ip o wood, a ound 10.0 cm ×1.2 cm ×1.2 cm (H ×W×D), was sawn angen ially om he hal s em disk (Figu e 2A). The s ip and he emaining s em piece we e immedia ely e u ned o liquid ni ogen. Du ing his i s s ep he ba k was easily emo ed along he expanding zone o he de eloping xylem, and hus, de eloping xylem wi h seconda y cell wall o ma ion on op o he ma u e xylem was al eady exposed o c yosec ioning. Du ing he second s ep, he ozen s ip o wood was immobilized in a ice and cubic pieces o ca. 1.2 cm ×1.2 cm ×1.2 cm we e sawn, placed in o 50 ml ubes in liquid ni ogen. The i s and he las cubes om he s ip we e disca ded, since a likely hawing in hese ou e mos zones o he s ip could comp omise he ana omy and he RNA in eg i y. The cubes we e s o ed a −80◦C, and used o c yosec ioning wi hin 7 days. De eloping xylem could be iden i ied on he angen ial side o he woody cube as a whi e, almos anslucen laye , as opposed o he yellowish, p e ious yea ’s g ow h ing. This acili a ed he o ien a ion o he cube du ing c yosec ioning. Al e na i ely, he side o he ma u e xylem in he cube was ma ked ed by a quick dipping in 0.5% Sa anin O (in 50% e hanol), be o e s o age a −80◦C. Aspen Aspen wood specimens we e collec ed om a 40-yea -old ee acco ding o he scheme illus a ed in Figu e 2B. The samples (ca. 30 cm ×20 cm ×5 cm) con aining ba k, cu en yea ’s g ow h and se e al olde annual g ow h ings, we e cu om he unk wi h a jigsaw, snap- ozen in liquid ni ogen and s o ed a −80◦C. Smalle wood blocks, app oxima ely o 3 cm ×1 cm ×3 cm, we e cu om he ozen wood ma e ial wi h a jigsaw, and kep in liquid ni ogen be o e immedia e c yosec ioning. Al e na i ely, hey we e s o ed a −80◦C in aluminum oil, o a longe pe iod. In aspen, he ba k was no emo ed, since i p o ec ed he F on ie s in Plan Science | www. on ie sin.o g 3Janua y 2017 | Volume 7 | A icle 1965 pls-07-01965 Decembe 27, 2016 Time: 12:11 # 4 Blokhina e al. Lase Cap u e Mic odissec ion P o ocol o Xylem FIGURE 1 | Flow cha o he c yosec ioning and mic odissec ion p o ocol. All he s ages o he p o ocol a e indica ed wi h he ela i e empe a u e and ime o ealiza ion. S eps pe o med di e en ly o No way sp uce and aspen a e, espec i ely, included in g een and magen a boxes. RT, oom empe a u e; o/n, o e nigh . de eloping xylem du ing he s o age. The ba k also helped o o ien cu ing du ing sec ioning. Key no es o s age A Fo collec ion o wood samples, di e en s a egies o cu ing can be applied (Figu e 2). The samples should be placed o liquid ni ogen as quickly as possible a e cu ing o smalle pieces o a oid hawing. We also ecommend o s o e he ozen wood specimen a −80◦C in small pieces sui able o a single expe imen . This minimizes he isk o hawing, and consequen ly limi s he isk o RNA deg ada ion. Du ing he p epa a ion o he small wood blocks, we ad ise o de ine a e e ence o easily iden i y he de eloping xylem zone, o example, by s aining as in he case o sp uce, o by keeping he ba k in place as we did o aspen. Equipmen and eagen s Jigsaw; plas ic bags; aluminum oil; 50 ml ubes; d y ice; liquid ni ogen; 0.5% Sa anin O in 50% e hanol). S age B. C yosec ioning Time: 1.5 h Tempe a u e:−20◦/−24◦C De ining he Di ec ion o Sec ioning The o ien a ion o he block de e mines he di ec ion o sec ioning (Figu e 3A). Table 1 summa izes ad an ages and disad an ages o p epa ing angen ial, c oss o adial sec ions. Tangen ial sec ions o e he bes iew o isola e di e en cell ypes, in pa icula ay cells (Figu e 3). Howe e , a c oss sec ion is necessa y o de ine he hickness o he de eloping xylem. In he case o aspen, he angen ial sec ions end o c ack along he longi udinal cell walls be ween ibe s and essels, and hence, c oss and adial sec ions we e p e e ed. P epa a ion o Tools o C yosec ioning Glass slides we e acid-washed in 1 M HCl o 16 h a c. 55◦C. A e se e al washing s eps in dH2O, he slides we e soaked in 100% e hanol, ai -d ied on a ack and s e ilized F on ie s in Plan Science | www. on ie sin.o g 4Janua y 2017 | Volume 7 | A icle 1965 pls-07-01965 Decembe 27, 2016 Time: 12:11 # 5 Blokhina e al. Lase Cap u e Mic odissec ion P o ocol o Xylem FIGURE 2 | Scheme o sampling o sp uce and aspen wood specimens. (A) Sp uce unk disk was sawn in hal es and hen, o con enience, in angen ial s ips o ca. 10.0 cm ×1.2 cm ×1.2 cm (1). Small blocks (2) we e cu om he s ips and used o c yosec ioning. (B) Aspen wood samples (1, 2) o ca 30 cm ×20 cm ×5 cm con aining ba k, cu en yea ’s g ow h and se e al olde annual g ow h ings, we e sawn o wood disks. (3) A small aspen wood block used o c yosec ioning. The g een dashed line ma ks he cu en yea ’s g ow h zone used o lase cap u e mic odissec ion. FIGURE 3 | Ana omy o a No way sp uce s em wi h c oss, adial and angen ial sec ions. (A) Schema ic ep esen a ion o he h ee sec ioning planes in a sp uce wood block. (B) Tangen ial sec ion s ained wi h Alcian blue and Sa anin O. CR, c oss sec ion; RA, adial sec ion; TA, angen ial sec ion. R, ay cell; B, ba k; P, phloem; C, ascula cambium; RD, esin duc ; TR, acheid. Scale ba 150 µm. a 180◦C o 4 h. Al e na i ely, he slides we e i s washed wi h RNase decon amina ion solu ion o 0.1% DEPC (die hyl py oca bona e)- ea ed wa e , hen wi h 100% e hanol and inally au ocla ed. As a hi d al e na i e ea men o emo e RNase con amina ion, he glass slides can also be incuba ed in a s e ile Pe i dish unde UV adia ion o 30 min. P io o c yosec ioning all he ools, including weeze s and azo blades, we e cleaned wi h he RNase decon amina ion solu ion. We do no ecommend he use o his solu ion o decon amina e he c yomic o ome, which can be cleaned wi h 100% e hanol ins ead. The empe a u e o he c yomic o ome was se o −20◦C, a leas 1 h be o e s a ing sec ioning. Du ing he i s hou o p e-cooling, moun ing disks and glass slides we e kep in s e ile Pe i dishes, and oge he wi h weeze s, azo blades, and e hanol solu ions cooled inside he c yomic o ome chambe . Memb ane-coa ed slides used in o he published p o ocols (Abbo e al., 2010;La isch e al., 2012;Zi hao e al., 2014), we e F on ie s in Plan Science | www. on ie sin.o g 5Janua y 2017 | Volume 7 | A icle 1965 pls-07-01965 Decembe 27, 2016 Time: 12:11 # 6 Blokhina e al. Lase Cap u e Mic odissec ion P o ocol o Xylem TABLE 1 | Choice o he di ec ion o sec ioning. Sec ion o ien a ion Ad an ages Disad an ages Tangen ial Easy isola ion o pu e ay cell lines. Easy o p epa e c yosec ions, as ibe s/ acheids go along he cu ing plane. Di icul o iden i y he bounda ies be ween he annual g ow h ings du ing sec ioning. A p elimina y es ima ion o he hickness o de eloping xylem is equi ed. In ha dwoods, he ligni ied cell walls o ibe s/ essels cause longi udinal c acks in he sec ion, and hampe he adequa e la ness o he sec ions on a glass slide. Radial Easy o iden i y ibe s and ay cells, and isola e hem in la ge amoun s. I he sec ion is pe ec ly adial (no oblique), he o ien a ion o ibe s/ acheids/ essels helps o keep he sec ion la . I he sec ion is no pe ec ly adial, he di ec ion o ibe s/ acheids/ essels makes i di icul o ix he sample on he slide and keep i la . Con amina ion om he su ounding ibe / acheid cells is no excluded. C oss Easy o dis inguish he de eloping xylem om he ma u e xylem, and o localize di e en ypes o cells. The po ous appea ance o he sec ion acili a es he lase cap u e mic odissec ion cu . When isola ing ay cells, con amina ion om he su ounding ibe / acheid cells canno be excluded. o iginally de eloped o issue sec ions o animals, and ha e been success ully adap ed o so plan issues. We disco e ed ha glass slides wo ked be e o de eloping xylem as discussed in mo e de ail unde “c i ical no es”. Es ima ion o he Thickness o De eloping Xylem in Sp uce The hickness o de eloping xylem in sp uce was de e mined using an ocula mic ome e om 20 µm hick c oss sec ions s ained wi h Sa anin O-Alcian blue solu ion (Figu e 4). The de eloping xylem laye used in ou s udy was app oxima ely 600 µm hick. Unligni ied cell walls s ain blue wi h Alcian blue, and pa ially ligni ied cell walls show some ed s aining wi h Sa anin O. Fully ligni ied cell walls om he p e ious yea ’s g ow h ing appea ed unde a b igh - ield mic oscope. P epa a ion o Tangen ial Sec ions o Sp uce The block o de eloping xylem was moun ed wi h weeze s on a cooled moun ing disk wi h he issue eezing medium. We i s pe o med a imming s ep, cu ing a se ies o 20 µm hick sec ions, o emo e abou 100 µm o he ou e mos laye o de eloping xylem. Then, 40 µm hick sec ions we e cu and placed on p e-cooled, RNase- ee glass slides. Each slide ca ied 3–4 sec ions. Acco ding o he p e ious es ima ion o he hickness o he de eloping xylem, he cu ing dep h did no exceed 600 µm. To acili a e he a achmen o he sec ions o he slide, he slide was sligh ly wa med om he back wi h a glo ed inge a he si e o he sec ions o ca. 10 s. This allowed collec ion o he eshly cu sec ions di ec ly om he c yomic o ome s age, and a oided any u he handling o damaging o he sec ions wi h weeze s. P epa a ion o C oss and Radial Sec ions o Aspen The ozen wood block was moun ed as desc ibed abo e o sp uce. Ba k and he oldes laye s o seconda y phloem we e emo ed by cu ing se ial, 20 µm hick angen ial sec ions. The younges laye s o he seconda y phloem, 500–700 µm om he cambium, we e le in ac o acili a e he c oss- and adial- sec ioning, and o p o ide a e e ence o he o ien a ion o he sec ions (Figu e 5). The measu emen o he hickness o he ba k and he de eloping xylem was done on c oss sec ions as desc ibed abo e o sp uce samples. C oss sec ions we e cu manually wi h a azo blade om he uppe side o he wood block du ing he imming s ep be o e changing he di ec ion o sec ioning. To check he co ec o ien a ion and posi ion o he wood sample on he c yo ome s age, he i s ew c yosec ions we e s ained wi h Sa anin O-Alcian blue, and iewed unde a b igh ield mic oscope (Figu e 5). Th ee o ou 40 µm hick c oss o adial sec ions (a ound 1cm×1 cm/1 cm ×1.5 cm) we e cu and delica ely posi ioned using s e ile weeze s in 50 µl o 75% ice-cold e hanol on a p e- cooled, RNase- ee mic oscope slide. The addi ion o e hanol helped he sec ions o adhe e o he slide. The glass slide was kep unco e ed and ans e ed in o a 50 ml ube, which was p e iously cooled in liquid ni ogen. The ube ca ying he slide was closed igh ly, snap- ozen in liquid ni ogen and kep on d y ice un il he eeze-d ying s ep. Key no es o s age B This s age o he p o ocol implies he longes handling ime o he samples be o e he ixa ion. Fo his eason, app op ia e measu es should be aken o limi he isk o RNA deg ada ion. Tools and equipmen need o be s e ile o ea ed wi h a RNase decon amina ion solu ion o , i no possible, wi h e hanol. Mo eo e , he whole p ocedu e has o be done quickly and a low empe a u e. We ecommend o keep all he ools, e.g., azo blades, weeze s, moun ing slides, in he c yomic o ome chambe . Equipmen and eagen s C yomic o ome (Leica CM3050 S); UV C osslinke (Spec olinke XL-1000); moun ing disks; eezing moun ing medium (Leica Tissue F eezing Medium, Re . 14020108926); mic oscope glass slides; glass Pe i dishes; 50 ml ubes; weeze s; azo blades; liquid ni ogen; RNaseZAPR decon amina ion F on ie s in Plan Science | www. on ie sin.o g 6Janua y 2017 | Volume 7 | A icle 1965 pls-07-01965 Decembe 27, 2016 Time: 12:11 # 7 Blokhina e al. Lase Cap u e Mic odissec ion P o ocol o Xylem FIGURE 4 | C oss sec ion o No way sp uce xylem o he es ima ion o he de eloping xylem hickness. A 20 µm hick c yosec ion was s ained wi h Sa anin O-Alcian Blue solu ion. Non-ligni ied cell walls in de eloping xylem s ain blue wi h Alcian blue ( he ou e pa o X2). Ligni ying cells ( he inne pa o X2) and he p e ious yea ’s g ow h ing (X1) wi h ully ligni ied cell walls s ain ed wi h Sa anin O. No e na ow iles o ay cells (a ows). P, phloem; C, cambium; X1, ma u e, p e ious yea ’s xylem; X2, cu en yea ’s xylem. Scale ba 250 µm. solu ion (AMBION AM9780); 100% e hanol; 1 M HCl; 0.1% DEPC (die hyl py oca bona e; Sigma–Ald ichR , 1609-47- 8)- ea ed wa e ; 75% e hanol solu ion in RNase- ee wa e (AMBION AM9930); solu ion (1:1, / ) o 0.5% Sa anin O(Me ck) in 50% e hanol and 0.5% Alcian blue (Na ional diagnos ics) in 50% e hanol. S age C. E hanol Fixa ion and Dehyd a ion o he C yosec ions Time: 20 min (sp uce); 30 min and o e nigh (aspen) Tempe a u e: oom empe a u e (sp uce); oom empe a u e and −50◦C (aspen) Fixa ion and D ying o he Sp uce Tangen ial Sec ions wi h E hanol Two minu es a e he las sec ion was placed on o he slide, ice- cold 70% e hanol was pipe ed in excess on op o he sec ions o ully co e he whole a ea. The slide was hen ans e ed in a s e ile glass Pe i dish in o a lamina low cabine , whe e 70% e hanol was eplaced wi h ice-cold 100% e hanol. The subsequen ixa ion s ep was pe o med a oom empe a u e in he lamina low cabine . Du ing he 2-min incuba ion in 100% e hanol, he sec ions we e gen ly s e ched and la ened wi h a pipe e ip (see c i ical no es). A e he emo al o e hanol by pipe ing, sec ions we e ai -d ied o 15 min. Slides wi h d y sec ions we e collec ed in o s e ile 50 ml ubes. The ubes we e igh ly closed and s o ed a −80◦C o no longe han 7 days. Fixa ion and Dehyd a ion by F eeze-D ying o Aspen Samples The eeze-d ye chambe was i s cleaned wi h he RNase decon amina ion solu ion and p e-cooled o −50◦C. The c yosec ions we e d ied as ollows: (i) in a closed 50 ml ube o 20 min. The ube cap was kep loose bu no o ally unsc ewed; (ii) in he o ally opened ube o addi ional 5–10 min; (iii) in a closed glass sandwich kep in he 50 ml ube, o e nigh . The glass sandwich was p epa ed by co e ing he glass mic oscope slide ca ying he sec ions wi h a second one and closing hem on hei sho sides wi h a pai o clips (Figu es 6A,B). Key no es o s age C This s age is pa icula ly impo an o ob ain la and d y sec ions equi ed o success ul LCM and good-quali y RNA ex ac ion (see c i ical no es). The glass sandwich keeps he specimens la du ing and a e d ying. Howe e , we obse ed ha he sec ions we e op imal o LCM i hey we e i s kep 30 min unco e ed in he eeze-d ye . Du ing his incuba ion, sec ions cu l and become pa ially de ached om he glass slide, bu a e s ill a ached all along he phloem and he cambium. A e his ini ial d ying s ep he sec ions can be la ened wi hou c acks by closing he glass sandwich o he ollowing o e nigh dehyd a ion. Equipmen and eagen s Sp uce: ice-cold 70% e hanol in RNase- ee wa e (QIAGEN); 100% e hanol; glass slides; pipe e ips; 50 ml ubes; lamina low cabine ; Aspen: eeze-d ye (Scan ac CoolSa eTM), RNaseZAPR  decon amina ion solu ion (AMBION AM9780); 50 ml ubes; mic oscope glass slides; o ice clips. S age D. Lase Cap u e Mic odissec ion Time: 1.5 h pe slide Tempe a u e: oom empe a u e Lase cap u e mic odissec ion s ep was pe o med simila ly o sp uce and aspen sec ions. The only di e ence was ha he aspen samples equi ed an addi ional p epa a o y s ep o immobilize he sec ions on glass slides. A he end o he o e nigh eeze-d ying, he glass sandwich was emo ed om he eeze-d ye and delica ely opened in he lamina low cabine . The d ied sec ions we e s ably a ached o he glass wi h s ips o ape on hei longe sides (Figu e 6C). The glass sandwich was closed again, using now only one clip o a oid F on ie s in Plan Science | www. on ie sin.o g 7Janua y 2017 | Volume 7 | A icle 1965 pls-07-01965 Decembe 27, 2016 Time: 12:11 # 8 Blokhina e al. Lase Cap u e Mic odissec ion P o ocol o Xylem FIGURE 5 | C oss sec ion o aspen xylem. A manual c oss sec ion o aspen wood was cu du ing he ini ial angen ial imming s ep o c yosec ioning. A ound 500–700 µm o he phloem (P) was le as a p o ec ion and o ien a ion e e ence du ing he p epa a ion o c oss/ adial aspen sec ions. C, cambial zone; X, cu en yea ’s xylem. Scale ba 200 µm. dis u bance and damage o he sec ions. Samples we e kep in s e ile Pe i dishes in a po able desicca o chambe , a oom empe a u e un il LCM. The sp uce samples we e anspo ed o he LCM appa a us in a 50 ml ube kep in liquid ni ogen. P io o LCM, he ube con aining he sp uce c yosec ions was kep a oom empe a u e o 20 min o p e en condensa ion on o he slide. The specimens we e dissec ed using he PALMR Mic oBeam Sys em (Ca l Zeiss AB). Op imal lase dissec ion was achie ed wi h bo h ypes o wood acco ding o he ollowing se ings: cu speed 20–30; cu ene gy 60–65; lase powe ca apul ing ene gy 25–30. The bes ocus was achie ed in he ange o 65–70 wi h del a ocus a –2. The op imal magni ica ion o ou lining and cu ing was 20×(sp uce) and 10×(aspen). This is simila o he mos sui able magni ica ion o ca apul ing (20× o sp uce and 10× o aspen) (Figu es 7 and 8). The dissec ed pieces we e collec ed by ca apul ing in o he adhesi e cap o a 500 µl collec ing ube. Each ube and each slide we e used du ing a single LMC session o no longe han 1.5 h. In his ime, we we e able o ca apul a ound 15–25 and 200–300 mic odissec ed pieces o aspen and No way sp uce, espec i ely, which gene ally co esponded o a o al a ea o 0.5–0.75 mm2 and 0.2–0.4 mm2o aspen and sp uce wood issue, espec i ely, collec ed in a single adhesi e cap. Specimens, dissec ed du ing se e al LCM sessions, we e combined o ob ain a de ec able amoun o ex ac ed RNA. Fo example, he o al a ea o sp uce LCM-isola ed ay pa enchymal cells was o ca. 12 mm2. A he end o e e y session (usually 1.5 h long), he collec ing ube was snap- ozen in liquid ni ogen. All he dissec ed samples we e s o ed a −80◦C un il RNA isola ion. Key No es We sugges o always p epa e mo e han one slide wi h 3–4 sec ions o one LCM session in o de o ha e enough sec ions in case one appea s unsui able o LCM. The use o RNase- ee ools ( weeze s and needles) du ing addi ion o ape o he eeze- d ied sec ions is ecommended al hough he sec ions a e now ixed, and hus, mo e p o ec ed agains RNA deg ada ion. In a oubleshoo ing Table 2, we lis he mos ele an and common p oblems aced du ing LCM. Equipmen and Reagen s Lase mic odissec ion mic oscope (PALM Mic oBeam, Zeiss); adhesi e cap ubes 500 µl opaque (Ca l Zeiss AB, 415190−9201−000); liquid ni ogen; 50 ml ubes; addi ionally o aspen: weeze s; needles; o ice anspa en ape (MagicTM in isible 3M, Sco chR ); scisso s; desicca o . S age E. RNA Isola ion and Assessmen o RNA Quali y Time: 2 h, o acco ding o he RNA ex ac ion ki manu ac u e ’s ins uc ions Tempe a u e: oom empe a u e and acco ding o he RNA ex ac ion ki manu ac u e ’s ins uc ions F on ie s in Plan Science | www. on ie sin.o g 8Janua y 2017 | Volume 7 | A icle 1965 pls-07-01965 Decembe 27, 2016 Time: 12:11 # 9 Blokhina e al. Lase Cap u e Mic odissec ion P o ocol o Xylem FIGURE 6 | Glass sandwich assembled o aspen sample p epa a ion. (A) The glass sandwich holding he woody c yosec ions is closed wi h a pai o o ice clips (B) o he o e nigh eeze-d ying p ocedu e. (C) The eeze-d ied sec ions a e a ached wi h ape on he moun ing slide and p o ec ed in a e-assembled glass sandwich closed by a single clip. In he close-up box, an example o d ied c yosec ion con aining an in ac xylem zone sui able o lase cap u e mic odissec ion. P, phloem; X1, old xylem; X2, cu en yea ’s xylem. FIGURE 7 | Lase cap u e mic odissec ion o ay cells om a angen ial sec ion o No way sp uce. (A) Ray pa enchymal cells a e ou lined (blue line) o lase cu ing. No e ha ay acheids (RT ) we e no collec ed. One cu and one uncu ile o ay cells a e seen. (B) Ray cells we e success ully cu and ca apul ed. Do s in he middle o he cu egions a e he a eas whe e he ca apul ing pulse was applied. T , acheid. Scale ba 150 µm. RNA was ex ac ed om he whole c yosec ions and dissec ed cells pooled om se e al LCM sessions. To al RNA quali y was assessed using he RNA Pico Assay o 2100 Bioanalyze . Aspen sec ions equi ed a longe p epa a ion ime han hose o sp uce, and we e consequen ly mo e suscep ible o RNA in eg i y impai men , especially du ing he las p e-LCM s age a oom empe a u e. To al RNA quali y was he e o e measu ed a e e y s ep o he p o ocol, i.e., in a single in ac c yosec ion, in a single in ac c yosec ion ixed and le a oom empe a u e in he desicca o o wo days, in mic odissec ed cells, and in esidual F on ie s in Plan Science | www. on ie sin.o g 9Janua y 2017 | Volume 7 | A icle 1965