Di ec con ocal acquisi ion o
luo escence om X-gal s aining on hick
issue sec ions
Kons an in L. Le i sky
1
, Juan Jose
´Toledo-A al
1,2,3
, Jose
´Lo
´pez-Ba neo
1,2,3
& Ja ie Villadiego
1,2,3
1
Ins i u o de Biomedicina de Se illa (IBiS), Hospi al Uni e si a io Vi gen del Rocı
´o/CSIC/Uni e sidad de Se illa, Se illa, Spain,
2
Depa amen o de Fisiologı
´aMe
´dica y Bio ı
´sica, Facul ad de Medicina, Uni e sidad de Se illa. Se illa, Spain,
3
Cen o de
In es igacio
´n Biome
´dica en Red sob e En e medades Neu odegene a i as (CIBERNED), Spain.
X-gal s aining is a common p ocedu e used in he his ochemical moni o ing o gene exp ession by ligh
mic oscopy. Howe e , his p ocedu e does no pe mi he di ec con ocal acquisi ion o images, hus
p e en ing he iden i ica ion o labelled cells on he dep h (Z) axis o issue sec ions and leading some imes
o e oneous conclusions in co-localiza ion and gene exp ession s udies. He e we epo a echnique, based
on X-gal luo escence emission and ma hema ically-based op ical co ec ion, o ob ain high quali y
luo escence con ocal images. This me hod, combined wi h immuno luo escence, makes i possible o
unequi ocally iden i y X-gal-labelled cells in issue sec ions, eme ging as a aluable ool in gene exp ession
and cell acing analysis.
Repo e genes a e used o moni o gene exp ession du ing emb yonic de elopmen and in he pos na al
pe iod. Among he di e en epo e sys ems, X-gal s aining (also e e ed o as LacZ s aining) has eme ged
as an op imal choice based on i s simplici y and high sensi i i y. This p ocedu e has been used o image
gene exp ession in a la ge ba e y o LacZ- used knockou mice as well as in he ROSA-26 s ain o C e- epo e
exp ession
1
. X-gal s aining is based on he p esence o he bac e ial LacZ gene ha encodes o he b-galac osidase
(b-gal) enzyme. The mos common subs a e used is 5-b omo-4-chlo o-3-indolyl b-D-galac opy aniside (X-gal),
which p oduces a da k blue p ecipi a e ha can be easily de ec ed by ligh mic oscopy
2–5
. Howe e , he use o ligh
ansmi ed mic oscopy o de ec X-gal s aining does no pe mi localiza ion o he p ecipi a e in a de ined
con ocal plane on issue sec ions. This makes i di icul o p ecisely de ine whe he o no a epo e gene is
exp essed in a pa icula cell wi hin a complex issue, despi e combining he de ec ion o he X-gal wi h immu-
nolabelling o o he cell ma ke s, p oducing a signi ican amoun o inco ec co-localiza ion on hick issue
sec ions. P e ious a emp s o ci cum en his majo limi a ion ha e been based on he use o speci ic an ibodies
agains b-gal
4
. Howe e , X-gal s aining is conside ably less cos ly and, as i is based on an enzyma ic eac ion,
o e s a lowe de ec ion h eshold and a be e signal o noise a io han immuno luo escence, which makes his
his ological me hod he i s -choice echnique o isualize low le els o gene exp ession. He e, we desc ibe a
simple me hod o ob ain high quali y luo escence con ocal images di ec ly om X-gal-s ained issue sec ions.
This echnique o di ec con ocal acquisi ion o luo escence om X-gal s aining is based on he ac ha he X-gal
p ecipi a e is able o abso b ligh in 570–700 nm ange, and emi luo escence in he 650–770 nm wa eleng h
ange. Impo an ly, he p ocedu e is o ally compa ible wi h classical immuno luo escence, allowing a combina-
ion o he LacZ epo e sys em wi h mul iple luo escen labels.
Resul s
To in es iga e he possibili y ha X-gal p ecipi a e can p oduce luo escence emission, we pu i ied X-gal om
LacZ-exp essing E.coli and analysed i s abso p ion and emission spec a. We de ec ed a peak o abso p ion in he
570–700 nm ange (Fig. 1A) and s udied whe he , a e abso p ion, he p ecipi a e could emi a luo escence
signal. Indeed, a e exci a ion o he X-gal p ecipi a e a 633 nm we de ec ed an emission signal in he 650–
770 nm ange (Fig. 1B). This inding makes i possible o ob ain a luo escence image using egula con ocal
mic oscopy. Figu e 1C shows an image o pu i ied X-gal p ecipi a e as seen by egula ligh mic oscopy, while
Fig. 1D illus a es images ob ained om he same pu i ied X-gal p ecipi a e isualized by ligh - ansmi ed
mic oscopy (le panel) and by luo escence con ocal mic oscopy ( igh panel) a e exci a ion a 633 nm, which
p oduces a high- esolu ion luo escence image ha was eco ded in he 650–770 nm wa eleng h ange. We did
OPEN
SUBJECT AREAS:
SUPER-RESOLUTION
MICROSCOPY
TECHNICAL REPORT
Recei ed
10 July 2013
Accep ed
27 Sep embe 2013
Published
14 Oc obe 2013
Co espondence and
eques s o ma e ials
should be add essed o
J.V. ([email p o ec ed])
SCIENTIFIC REPORTS | 3 : 2937 | DOI: 10.1038/s ep02937 1
no de ec any luo escen signal om he same X-gal p ecipi a e wi h
exci a ion wa eleng hs anging om 405 o 561 nm (Supplemen a y
Fig. 1).
The p ocedu e desc ibed abo e (exci a ion a 633 nm and eco d-
ing luo escence emission in he 650–770 nm ange) was subse-
quen ly es ed on X-gal-s ained issue sec ions. Figu e 2A shows
wo X-gal p ecipi a es on a 30 mm hick b ain sec ion om a
GDNF/X-gal
6,7
mouse isualized by egula ligh mic oscopy (le
panel) and by ligh ansmission mic oscopy ( igh panel) a e illu-
mina ion wi h a Helium-Neon 633 nm lase . Al hough bo h X-gal
p ecipi a es we e clea ly de ec ed by ligh mic oscopy, his app oach
does no pe mi localiza ion o he p ecipi a e in a de ined con ocal
plane in he dep h (Z) axis. We exci ed he issue sec ion a 633 nm
and eco ded emi ed luo escence 650–770 nm ange o a Z-s ack
(7.56 mm) ob aining con ocal images ha e ealed wo luo escen
do s pe ec ly coinciden wi h he X-gal p ecipi a es bu in wo
Figu e 1
|
X-gal luo escence emission a e exci a ion a 633 nm. (A). Abso p ion spec um o X-gal. No e he peak o abso p ion in he 570–700 nm
ange. l
abs
5abso p ion ligh wa eleng h in nanome e s. (B). Fluo escence emission spec um o X-gal a e exci a ion a 633 nm showing
luo escence emission in he 650–770 nm ange; a.u: a bi a y uni s, l
em
5emi ed ligh wa eleng h in nanome e s. (C). Ligh mic oscopy image om
pu i ied X-gal p ecipi a e deposi ed on a co e slip. (D). Images ob ained om he same pu i ied X-gal p ecipi a e showed in C isualized by ligh
ansmi ed mic oscopy (le panel) and by luo escence con ocal mic oscopy ( igh panel) a e exci a ion wi h a wa eleng h o 633 nm.
Figu e 2
|
Con ocal acquisi ion o luo escence om X-gal s aining on hick issue sec ions. (A). X-gal inclusions in a b ain co onal sec ion om a
GDNF/LacZ mouse, isualized using egula ligh mic oscopy (le panel) o by ligh ansmission mic oscopy a e illumina ion a 633 nm in a con ocal
mic oscope ( igh panel). (B). Con ocal acquisi ion o luo escence ( om a 7.56 mm Z-s ack, oxel dep h o 0.12 mm and pinhole o 1 ai y) o he X-gal
inclusions in A a e exci a ion a 633 nm. Al hough bo h X-gal p ecipi a es seem o appea in he same Z-plane in A (ligh mic oscopy), hey a e ac ually
loca ed in comple ely di e en Z-planes (z 514 in le panel and z 548 igh panel; see scale o ansmi ance and luo escence alues in A and B).
(C). Images shown in B a e applica ion o he ma hema ically-based op ical co ec ion p ocedu e o elimina e backg ound luo escence: sub ac ion o
he ansmi ance alues a e exci a ion a 633 nm om he luo escence digi al image. (D,E). C oss-sec ion o he p e iously shown con ocal-z-planes
(z 514 in D; and z 548 in E) illus a ing he x-y-z coincidence o he luo escence signal (le panels) wi h he X-gal inclusion ( igh panels, isualized by
ligh ansmission mic oscopy).
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SCIENTIFIC REPORTS | 3 : 2937 | DOI: 10.1038/s ep02937 2
di e en dep h (Z) axis planes sepa a ed by ,4mm [Fig 2B; Z 514
in he le panel and Z 548 in he igh panel; oxel dep h o 0.12 mm
(Z-dep h esolu ion o con ocal planes) and pinhole 1 ai y uni ].
Al hough exci a ion o X-gal deposi s allows he acquisi ion o a
luo escence signal ha is clea ly coinciden wi h he X-gal p ecip-
i a e, i p oduces a signi ican le el o backg ound luo escence in he
non-X-gal-labelled su ounding issue (no e he small luo escence
signals no coinciden wi h he X-gal p ecipi a es in Fig. 2B). This
backg ound signal, some imes appea ing as de ined s uc u es, could
be due o au o- luo escence o he neu al issue in he eco ded
wa eleng h ange. Indeed, au o- luo escence emission, on his ange
wa eleng h, by lysosomal lipo uscin has been epo ed
8
. Backg ound
noise could be inc eased by he high gain o he pho omul iplie
necessa y o acqui e he weak luo escence emission om he X-
gal p ecipi a es. To imp o e he signal o noise a io, we designed a
ma hema ically-based op ical co ec ion me hod ha signi ican ly
educes he backg ound luo escence. This p ocedu e consis s o
he simul aneous eco ding o he luo escence and ligh ansmi ed
signals by hei espec i e pho omul iplie s once he issue sec ion
has been exci ed a 633 nm. Bo h he luo escence and ligh ans-
mi ed images a e digi ized using, o ins ance, an 8-bi scale (see
scale alues in Fig. 2A and B). The digi ized alues o ansmi ance
a e hen sub ac ed om he luo escence image alues, esul ing in
high- esolu ion con ocal images o luo escence om he X-gal
inclusions (Fig. 2C; le , Z 514 and igh Z 548). Since X-gal
p ecipi a es a e opaque, wi h ansmi ance alues close o 0 ( ull
abso bance), and he su ounding biological issue is su icien ly
clea o p o ide high alues o ansmi ance (see ela i e scale o
ansmi ance alues showed in Fig. 2A), his co ec ion p ocedu e
signi ican ly educes he le el o he non-speci ic signal om he
non-X-gal-labelled issue wi hou an accompanying loss o signal
om he p ecipi a e. Impo an ly, he ansmi ance alues should
be acqui ed a e exci a ion o he sample a 633 nm because his
wa eleng h p oduces he mos opaque image o he X-gal p ecipi a e
om he di e en wa eleng hs es ed. Thus, highe di e ences in he
ansmi ance alues be ween he opaque X-gal inclusions and he
clea , non-labelled issue, a e ob ained (see Fig. 1D ansmi ed ligh
panel). The combina ion o he X-gal luo escence emission wi h he
op ical co ec ion me hod allows one o ob ain high quali y X-gal
luo escence con ocal images which a e pe ec ly coinciden wi h he
X-gal inclusion (Fig. 2D,E), hus making possible he unequi ocal
localiza ion o he X-gal p ecipi a e in a de ini e x-y-z posi ion on
hick issue sec ions.
The desc ibed p ocedu e is pa icula ly ele an o analysing sec-
ions om complex issues composed o di e en cell ypes ha may
be in close con ac . A signi ican ad an age o he di ec luo escen
con ocal acquisi ion o X-gal s aining me hod is i s compa ibili y wi h
he emission ange (,400–620 nm) o mos common luo opho es
used in immunohis ochemical s udies, hence making possible he
combina ion o he LacZ epo e sys em wi h o he an igens o luo -
escen dyes. To u he illus a e he pe o mance o his new
app oach, we applied he con ocal X-gal luo escence de ec ion o
30 mm- hick neos ia al sec ions om a GDNF/X-gal mouse; he sec-
ions we e labelled wi h ma ke s o in e neu ons (pa albumin-Alexa
Figu e 3
|
Compa ibili y o he con ocal acquisi ion o luo escence om X-gal s aining wi h mul iple luo escence labeling. (A). T ansmi ance image
a e illumina ion a 633 nm (le panel) and he maximal luo escen p ojec ion images om X-gal (cen al panel) and me ged luo escence (X-gal/
DAPI/GFAP/pa albumin; igh panel) ob ained by con ocal acquisi ion o a Z-s ack o a 25.4 mm- hick o a GDNF/LacZ b ain sec ion a e X-gal
s aining and immuno luo escence. (B–D). C oss-sec ions o he con ocal-z-planes ( oxel dep h o 0.488 mm) in which each X-gal luo escen do in F
(ma ked by a ows; igh panel) a e loca ed.
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SCIENTIFIC REPORTS | 3 : 2937 | DOI: 10.1038/s ep02937 3
568 nm exci a ion wa eleng h), as ocy es (glial ib illa y acid p o ein,
GFAP-Alexa 488 nm exci a ion wa eleng h) and nuclei (49,69-diami-
dino-2-phenylindole, DAPI). Figu e 3A shows he ansmi ance
image a e exci a ion a 633 nm (le panel) oge he wi h he max-
imal p ojec ion images o X-gal (cen al panel) and me ged luo -
escence (X-gal/DAPI/GFAP/pa albumin; igh panel) ob ained by
con ocal acquisi ion (25.4 mm Z-s ack, 0.48 mm oxel dep h and pin-
hole o 1 ai y). The me ged luo escence image (Fig. 3A, igh panel)
e eals X-gal luo escence do s (a ows B, C and D) ha , as shown in a
p e ious epo
9
, seem o be inside pa albumin-posi i e neu ons, in
e y close con ac wi h GFAP-posi i e glial p ocesses (a ows B and
C), o e en ou side o he neu onal soma (a ow D). De ailed inspec-
ion o he Z-con ocal planes co esponding o each X-gal luo escence
do e ealed unequi ocally ha he X-gal deposi s a e p oduced by
pa albumin-posi i e neu ons (Fig. 3B–D). No ably, X-gal luo -
escence do s shown in Fig. 3C,D a e loca ed wi hin he same neu on
bu appea in di e en z-con ocal planes (z 522 in Fig. 3C and z 525
in Fig. 3D; sepa a ed in he dep h (Z) axis by ,1.5 mm; see also
Supplemen a y Fig. 2 illus a ing he X-gal image p ocessing ope a-
ions). These images e eal ha ou me hod no only has su icien
esolu ion o localize he X-gal p ecipi a e in a pa icula cell bu also
wi hin subcellula s uc u es o hick issue sec ions. No e ha his
echnique is pa icula ly use ul o deciphe e oneous cases o appa -
en co-localiza ion o X-gal p ecipi a es in hick issue sec ions.
Figu e 4A–C shows a ypical analysis o co-localiza ion by classical
ligh ansmi ed/ luo escence mic oscopy o a co onal b ain sec ion
a e X-gal s aining, DARPP32 immuno luo escence and nuclea
s aining wi h DAPI. Blue X-gal do s (labelled by a owheads) seem
o appea wi hin a medium spiny, DARPP32-posi i e, cell, which is
he neu onal class mos abundan in he neos ia um. Howe e , he
analysis o he c oss-sec ions o speci ic con ocal-z-planes (Fig. 4D, z
515; Fig. 4E, z 519 and Fig. 4F, z 58; and Supplemen a y Fig. 3
showing he X-gal image p ocessing ope a ions) o he same egion
using ou me hod clea ly e eals he absence o co-localiza ion
be ween he DARPP32-posi i e neu ons and he X-gal do s. The e-
o e ou p ocedu e o di ec con ocal acquisi ion o luo escence om
X-gal s aining is c i ical o a oid misin e p e a ion o classical ligh
ansmi ed/ luo escence mic oscopy images due o he impossibili y
o localizing he X-gal signal in a de ined dep h (Z) plane.
Images shown in p e ious igu es we e ob ained om he GDNF/
X-gal mouse line, in which he exp ession o he b-gal enzyme is
unde he con ol o he endogenous GDNF p omo e . This epo e
mouse line shows a cha ac e is ic X-gal s aining on neu al issue,
p esen ing disc e e X-gal deposi s due o he low le el o exp ession
o he b-gal enzyme
7,9–11
. We also es ed he alidi y o ou me hod in
a high-exp ession LacZ epo e line using a TH-i es-C e/R26R
mouse (ca ying he Rosa26R-LacZ epo e cons uc ). Figu e 5A,B
shows a co onal b ain sec ion om a TH-i es-C e/R26R mouse a e
Figu e 4
|
Compa ison be ween ligh ansmi ed/ luo escence mic oscopy and di ec luo escen con ocal acquisi ion in he analysis o complex and
misleading X-gal op ical images. A–C. Ligh ansmi ed (A), luo escence (B) and he me ged image (C) o a co onal b ain sec ion a e X-gal s aining,
DARPP32 immuno luo escence and nuclea s aining wi h DAPI. Blue X-gal do s (labelled by a owheads) seem o appea coinciden wi h a DARPP32-
posi i e neu on. (D–F). C oss-sec ions o speci ic con ocal-z-planes (D, z 515; E, z 519 and F, z 58) o he same egion analysed in A–C,
e ealing he absence o co-localiza ion be ween he DARPP32-posi i e neu ons and he X-gal do s. No e he la e al iews a e 3D- econs uc ion,
adjacen o panel F, showing ha he luo escen X-gal do s do no co espond wi h any DARPP32-posi i e neu on. Con ocal acquisi ion om a 20.0 mm
Z-s ack, oxel dep h o 0.8 mm and pinhole ai y 1.
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SCIENTIFIC REPORTS | 3 : 2937 | DOI: 10.1038/s ep02937 4
X-gal s aining and isualiza ion by classical ligh ansmi ed mic o-
scopy (Fig. 5A) and 633 nm illumina ion (Fig. 5B). As expec ed, his
LacZ epo e line shows high le els o X-gal labelling dis ibu ed
h oughou he soma o mesencephalic dopamine gic TH
1
neu ons
(Fig. 5A,B,D). In addi ion, some dispe se X-gal inclusions also
appea ed as spa se do s ha could be a ibu ed o low le els o b-
gal ac i i y on TH
1
neu onal p ocesses (see Fig. 5D). The con ocal
images showed in Fig. 5C,D clea ly illus a e ha he luo escen X-
gal signal a e coinciden wi h he X-gal s aining e ealed by classical
ligh ansmi ed mic oscopy (Fig. 5A,B,D; and Supplemen a y Fig. 4
showing he X-gal image p ocessing ope a ions). Mo eo e , he luo -
escen con ocal X-gal label pe ec ly ma ches wi h he soma o TH
1
neu ons as e ealed by TH immuno luo escence (Fig. 5E–I). In e es -
ingly, high magni ica ion con ocal images epo ed in Fig. 5G,I indi-
ca ed ha he dispe se X-gal inclusions we e inside dopamine gic
TH
1
neu onal ib es. These expe imen s illus a e ha he di ec
con ocal acquisi ion o luo escence om X-gal s aining could be
pe o med on cells o issues wi h high le el o b-gal ac i i y, acil-
i a ing he co-localiza ion analysis by he use o mul iple luo escen
labelling. Mo eo e , his me hod eme ges as an excellen ool o
localize and clea ly iden i y cells o subcellula s uc u es ha p esen
low le els o b-gal exp ession o ac i i y.
Discussion
In his pape we epo a simple echnique o pe o m di ec con ocal
acquisi ion o luo escence om X-gal s aining. The possibili y o
ob ain con ocal images om X-gal s aining has been p e iously
epo ed by using nea -in a ed pho oac i a ion (720–730 nm) o
he X-gal p ecipi a e wi h a Ti anium-doped Saphi e (Ti:S) lase
12
.
Howe e , he me hod p esen ed he e, combined wi h ma hema -
ically-based op ical co ec ion, allows high quali y con ocal images
o be ob ained om he X-gal-s ained sec ions wi h he aid o a
egula con ocal mic oscope ( i ed wi h a Helium-Neon 633 nm
lase ) wi hou pho oac i a ion, being o ally compa ible wi h he
use o mos common luo escen dyes o immunolabelling. As pa
o ou me hodology we ha e also designed a ma hema ically-based
op ical co ec ion p o ocol, which is he i s desc ip ion o a eliable
me hod o signi ican ly inc ease he signal o noise a io o weak
luo escence.
The LacZ gene is widely used as a epo e sys em in di e en
o ganisms, including a b oad spec um o gene ically modi ied mouse
lines. He e, we ha e p esen ed an easy o implemen and p ac ical
p ocedu e o pe o m he con ocal acquisi ion o luo escence om
X-gal-s ained sec ions, which sol es a majo limi a ion o his
epo e sys em. The new me hodology unequi ocally localizes he
X-gal p ecipi a es in de ined dep h (Z) ocal planes and can be com-
bined wi h mul iple luo escen labelling, hus alluding o i s use as a
aluable ool in gene exp ession and cell acing s udies. In e es ingly,
since senescence-associa ed be a-galac osidase ac i i y has been
widely used as bioma ke o senescen cells
13
, he me hod desc ibed
he e could be also applied o e alua e cell senescence in issue o
umo sec ions. In addi ion, he luo escen p ope y o X-gal makes
he epo e a good candida e o new, and e en mo e in e es ing,
applica ions such as low cy ome y o dynamic luo ome y.
Figu e 5
|
Fluo escence con ocal acquisi ion om X-gal s aining using he high LacZ exp ession TH-i es-C e/R26R epo e mouse line. (A–C), B ain
co onal sec ion o a TH-i es-C e/R26R mouse showing high le el o X-gal s aining on he dopamine gic mesencephalic neu ons, isualized by classical
ligh ansmi ed mic oscopy (A), ansmi ed mic oscopy a e 633 nm illumina ion (B) and by luo escence X-gal con ocal acquisi ion (C). (D–F),
images co esponding wi h he inse ma ked in B, showing he ansmi ance (D, a e 633 nm illumina ion), X-gal luo escence (E) and X-gal/TH
luo escence me ged signal (D, a e TH inmu luo escence). (G–I), High magni ica ion pic u es o he a ea labelled by he inse in panel E, illus a ing he
X-gal luo escence (G) and DAPI/TH (H) o X-gal/TH (I) me ged images. Images epo ed in B and C we e acqui ed using he mosaic unc ion (wi h a
203objec i e) o he con ocal mic oscope. Con ocal adquisi ion om a 12.0 mm Z-s ack, oxel dep h o 0.498 mm and pinhole ai y 1.23.
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SCIENTIFIC REPORTS | 3 : 2937 | DOI: 10.1038/s ep02937 5

Me hods
Animals.Mos o he expe imen s we e ca ied ou wi h he e ozygous GDNF/LacZ
mice
6,7
. In addi ion, con ocal acquisi ion o luo escence om X-gal s aining was also
pe o med on TH-i es-C e/R26R mouse. All expe imen s we e pe o med acco ding
o he animal ca e guidelines o he Council o he Eu opean Union (86/609/EEC),
and we e app o ed by he Animal Resea ch Commi ee o he Uni e si y Hospi al
Vi gen del Rocı
´o (Uni e si y o Se ille).
Pu i ica ion and abso p ion/emission spec a o X-gal.E. coli (DH5a) exp essing
b-galac osidase and con ols (wi hou b-galac osidase exp ession) we e g own
o e nigh a 37uC in Lu ia b o h media (In i ogen) wi h 100 mg/ml ampicillin,
0.5 mM isop opyl-b-D-1- hiogalac opy anoside (IPTG) and 80 mg/ml X-gal.
Bac e ia we e ha es ed by cen i uga ion a 16000 g o 2 min and we e lysed wi h P1
lysis bu e (Quiagen). Cell ex ac s we e cen i uged a 16000 3g o 2 min, he
supe na an s we e disca ded and he blue p ecipi a e was collec ed, esuspended in
70% ( / ) pe chlo ic acid and incuba ed a 22uC o 15 min. The ea e , he
suspensions we e cen i uged a 16000 g o 5 min a 4uC, he p ecipi a e suspended
in PBS and deposi ed on a co e slip, and images ob ained as shown in Fig. 1C and D.
The emission spec um was also ob ained by a lscan me hod on a Leica TCS SP2
con ocal mic oscope (Fig. 1B). To ob ain he abso p ion spec um, he X-gal
p ecipi a e was dissol ed wi h 0.1 N NaOH and analysed on an ul a iole - isible
spec opho ome e (Beckman DU 640; Beckman), wi h he suspension ob ained om
he con ol bac e ia (wi hou b-galac osidase exp ession) used as blank.
His ology.A e ansca dial pe usion o mice wi h 50 ml o PBS (Sigma) and 50 ml
o 4% pa a o maldehyde (Sigma) in PBS a 37uC, b ains we e immedia ely emo ed
and ixed o 2–3 hou s a 4uC wi h 4% pa a o maldehyde in PBS. Co onal sec ions
( hickness 30–50 mm) we e cu wi h a c yos a (Leica) o ib a ome (Leica). X-gal
s aining was pe o med on sec ions om GDNF/Lacz he e ozygous mice
6,7
o TH-
i es-C e/R26R mice. B ie ly, b ain co onal sec ions we e washed 3 imes in solu ion C
(137 mM NaCl, 2.7 mM KCl, 10 mM Na
2
HPO
4
, 2 mM KH
2
PO
4
, 2 mM MgCl
2
,
5 mM EGTA, 0.02% IGEPAL, 0.01% Na desoxychola e) and incuba ed o e nigh on
X-gal s aining solu ion (solu ion C 110 mM K
3
FeCN
6
,10 mMK
4
FeCN
6
, 0.5 mg/ml
X-gal). A e X-gal s aining, immuno luo escence de ec ion was pe o med as
p e iously desc ibed
7
o glial ib illa y acid p o ein (GFAP; 15500; Dako),
pa albumin (155000, Swan ) and DARPP32 (15200, Millipo e). An i-mouse IgG
conjuga ed wi h Alexa Fluo 488 o an i- abbi IgG conjuga ed wi h Alexa Fluo 568
(15400; In i ogen) we e used as seconda y an ibodies. Nuclei we e s ained wi h
DAPI (151000; Sigma). B ain sec ions we e moun ed wi h Dako luo escence
moun ing medium (Dako) o Vec ashield luo escence moun ing medium (Vec o ).
T ansmi ed ligh images (le panel in Figu e 2A) we e ob ained using an Olympus
AX70 mic oscope wi h a DP72 e ige a ed digi al came a. Con ocal images we e
acqui ed using a Leica TCS SP2 con ocal mic oscope.
Con ocal acquisi ion and op ic co ec ion me hod.Di ec con ocal acquisi ion o
luo escence was pe o med using a Leica TCS SP2 mic oscope (Leica) equipped wi h
a Blue-diode 405 nm, A gon-K yp on 458–514 nm, Helium-Neon 543 nm, and
Helium-Neon 633 nm lase s. Mos o he images we e acqui ed using a HCX Pl Apo
203/0.70 o HCX Pl Apo CS 633/1.3 imme sion objec i es. Tissue sec ions we e
exci ed a 633 nm wi h he Helium-Neon 633 nm lase , and he ansmi ance signal
and he luo escence signal ( eco ded a 650–770 nm) we e isualized o adjus he
gain o he luo escence pho omul iplie un il a clea luo escence signal coinciden
wi h he X-gal p ecipi a es (which a e simul aneously isualized ia he ligh
ansmi ed channel) was ob ained. Once he op imal gain o he luo escence
pho omul iplie had been de e mined, bo h he luo escence emission (650–770 nm)
and he ansmi ance signal we e eco ded on a speci ic Z-s ack. Fluo escence and
ansmi ed ligh images we e digi ized wi h an 8-bi scale and hei alues we e
ob ained wi h he app op ia e con ocal mic oscope so wa e ools (Leica TCS SP2).
The ansmi ance alues we e sub ac ed om he luo escence images on each
con ocal plane, and he esul ing X-gal luo escence images ( ansmi ance
sub ac ed) we e s o ed. The blu il e (3 33) was applied o mos o ou X-gal
luo escen images. This image p ocessing il e s he high equencies ou o he
image, educing he dis inc i e ansi ions om low o high in ensi y alues
(Supplemen a y Fig. 2–4 show a comple e se o images illus a ing he whole X-gal
luo escence image p ocessing). The ea e , con ocal acquisi ion o he addi ional
luo escence labels was pe o med as ollows: DAPI (exci ed a 405 nm and eco ded
on 415–450 nm), GFAP-Alexa 488 nm (exci ed a 488 nm and eco ded a 515–
550 nm), and pa albumin-Alexa 568 nm (exci ed a 561 nm and eco ded a 575–
620 nm).
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Acknowledgmen s
We hank A.M. Mun
˜oz-Cabello o c i ical eading o he manusc ip , o J.I. Pi ua and B.
Dı
´az-Cas o o p o iding his ological sec ions and images, and N. Sua ez-Luna and A.
Be mejo-Na as o help wi h he his ology. Suppo was ob ained om he Spanish
Minis ies o Economy and Inno a ion (SAF, FIS, Red TERCEL, and CIBERNED
p og ams), he Andalusian Go e nmen , and he Bo ı
´n Founda ion.
Au ho con ibu ions
K.L.L. and J.V. designed and pe o med he expe imen s. J.T.A. and J.L.B. supe ised he
p ojec . J.V. and J.L.B. w o e he manusc ip .
Addi ional in o ma ion
Supplemen a y in o ma ion accompanies his pape a h p://www.na u e.com/
scien i ic epo s
Compe ing inancial in e es s: The au ho s decla e no compe ing inancial in e es s.
How o ci e his a icle: Le i sky, K.L., Toledo-A al, J.J., Lo
´pez-Ba neo, J. & Villadiego, J.
Di ec con ocal acquisi ion o luo escence om X-gal s aining on hick issue sec ions. Sci.
Rep.
3
, 2937; DOI:10.1038/s ep02937 (2013).
This wo k is licensed unde a C ea i e Commons A ibu ion-
NonComme cial-Sha eAlike 3.0 Unpo ed license. To iew a copy o his license,
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SCIENTIFIC REPORTS | 3 : 2937 | DOI: 10.1038/s ep02937 6