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5837 Microchip Implant
5837 Microchip Implant
Toolbox
Editor’s Note: Toolboxes are intended to briefly highlight a new method or a resource of general use in neuroscience or to critically
analyze existing approaches or methods. For more information, see http://www.jneurosci.org/misc/itoa.shtml.
Visualizing the proliferation marker bro- modeoxyuridine (BrdU) into replicating HCl pretreatment, pretreatment by incu-
modeoxyuridine (BrdU) requires pre- DNA and the subsequent immunohisto- bating fixed tissue with sodium citrate
treatment of tissue, typically with dilute chemical detection of the BrdU (Gratzner, buffer heated to near 100°C in an ordinary
hydrochloric acid (HCl). We report here 1982; Miller and Nowakowski, 1988). Ad- rice/vegetable steamer results in brighter
that pretreatment by steam heating of ministration of BrdU to living animals or fluorescent labeling of cells that have in-
paraformaldehyde-fixed tissue sections addition of BrdU to culture medium can corporated BrdU and at the same time al-
covered with citrate buffer yields much be used to determine relative proliferation lows high-quality labeling of nuclear DNA
brighter labeling of BrdU than HCl pre- rates, the length of the cell cycle, and the and various additional antigens. Although
treatment, allows labeling with many an- percentage of cells in the cell cycle (growth similar protocols for preparation of tissue
tibodies greatly superior to HCl pretreat- fraction). In BrdU-labeling experiments, for BrdU labeling have been reported pre-
ment, and allows concurrent high-quality it is often desirable to label various anti- viously (Dover and Patel, 1994), others
labeling of nuclei with the DNA-binding gens in addition to BrdU; such additional have found this type of pretreatment inef-
dyes Hoechst, DAPI (4⬘,6-diamidino-2- labels might, for example, be used to de- fective (Valero et al., 2005), and HCl pre-
phenylindole), and Syto24. Standard use termine whether the proliferating cells ex- treatment continues to be the standard.
of antigen retrieval by steamer can facili- press antigens characteristic of a particu- We discuss here critical parameters that
tate new insights into mechanisms regu- lar cell-type identity (Menezes and we have identified for using hot citrate
lating normal progenitor and tumor cell Luskin, 1994) and/or to determine total buffer pretreatment and describe a robust
proliferation and novel understandings of cell number to calculate the percentage of protocol for this method.
protein expression through increased sen- BrdU-labeled cells.
sitivity of immunohistochemical analysis. Antibody detection of BrdU incorpo- Materials and Methods
Equipment and reagents
rated into DNA requires pretreatment of The steamer unit used is described below.
Introduction the tissue or cells to expose the BrdU Chemicals used included the following: boric
A method commonly used to label cells in epitope. Commonly, this “antigen re- acid (catalog #BP168; Fisher, Pittsburgh, PA),
the S-phase of the cell cycle involves in- trieval” is done by incubating the slides in BrdU (catalog #B5002; Sigma, St. Louis, MO),
corporation of the thymidine analog bro- citric acid (catalog #C0759; Sigma), HCl (12.1
warm 2.0 M hydrochloric acid (HCl).
M; catalog #A144; Fisher), sodium chloride
However, such treatment abolishes spe-
Received Nov. 21, 2006; revised April 12, 2007; accepted April 19, 2007. (NaCl; catalog #S271; Fisher), sodium hydrox-
cific labeling by dyes binding to double-
This work was supported by National Institute on Deafness and Other ide (NaOH; catalog #S320; Fisher), sodium
Communication Disorders Grant RO1 DC03190 (M.B.L.). We thank Julie stranded DNA, such as Hoechst 33342, phosphate dibasic anhydrous (Na2HPO4; cata-
Siegenthaler and Dr. Michael Miller (State University of New York, Syra- Syto 24, and 4⬘,6-diamidino-2- log #S374; Fisher), sodium phosphate mono-
cuse, NY) for discussions. phenylindole (DAPI), markers that are basic monohydrate (NaH2PO4䡠H2O; catalog
*X.T. and D.L.F. contributed equally to this work. commonly used for counting total cell #BP330; Fisher), sucrose (catalog #BP220;
Correspondence should be addressed to either of the following: Dr.
Marla B. Luskin, Department of Cell Biology, Whitehead Biomedical Re-
number. Furthermore, this HCl pretreat- Fisher), sodium tetraborate decahydrate (cata-
search Building, Room 548, Emory University School of Medicine, 615 Mi- ment substantially reduces or abolishes log #B9876; Sigma), and Tris base (catalog
chael Street, Atlanta, GA 30322, E-mail: luskin@cellbio.emory.edu; or Dr. specific labeling by many antibodies, #BP152; Fisher). Antibodies and DNA-binding
Douglas L. Falls, Department of Cell Biology, Whitehead Biomedical Re- thereby compromising determination of dyes used are listed in Table 1.
search Building, Room 546, Emory University School of Medicine, 615 Mi-
chael Street, Atlanta, GA 30322, E-mail: dfalls@emory.edu.
codistribution of BrdU incorporation Buffers
DOI:10.1523/JNEUROSCI.5048-06.2007 with various proteins of interest. Citric acid. The standard buffer used in our
Copyright©2007SocietyforNeuroscience 0270-6474/07/275837-08$15.00/0 We report here that, compared with protocol is 10 mM sodium citrate, pH 6.0, pre-
5838 • J. Neurosci., May 30, 2007 • 27(22):5837–5844 Tang et al. • Toolbox
pared by diluting a 1.0 M, pH 6.0 stock. The 1.0 slides were exposed. After filling the steamer to the desired temperature, the slides were im-
M citric acid, pH 6.0 stock was prepared as fol- reservoir with clean water, the covered lower mersed in the buffer in the Coplin jar and in-
lows: 48.03 g of citric acid was dissolved in 150 steaming bowl containing the tube rack was cubated in the water bath for 15 min. At the
ml of water. Water was added to increase the placed on the steamer base, cooking time end of the incubation, immunohistochemical
volume to ⬇180 ml. The pH was adjusted to was set to ⬎45 min, and the “On” button was processing was initiated beginning with a PBS
6.0 with 10.0 M NaOH (⬇60 ml). Water was pressed to start heating. The temperature in the wash of the slides followed by application of
added to bring the final volume to 250 ml, the steamer bowl must be ⬇99°C during the entire blocking solution as described below. For our
pH was rechecked, and an additional adjust- tissue incubation time; achieving this required water bath, when the thermostat was set to
ment made, if necessary. For 10 mM sodium preheating the steamer for ⬇10 –15 min. Slides 100°C, the temperature of the equilibrated
citrate, pH 6.0, the 1.0 M stock was diluted were washed in PBS three times for 10 min and buffer was 97°C; when set to 95°C, the temper-
1:100 with water. For experiments assessing the then placed on the rack in the steamer, and the ature was 95°C; when set to 90°C, the temper-
effect of citrate concentration on labeling in- tissue was covered with buffer solution (800 ature was 90°C.
tensity, we also prepared 100 mM sodium ci- l/slide) gently applied using a pipettor. (The Microwave. A 700 W Kenmore brand micro-
trate, pH 6.0 by diluting the 1.0 M stock 1:10 standard buffer is 10 mM sodium citrate, pH wave oven (Model 721) was used. We designed
with water. For citrate and other buffers used, 6.0, but other buffers were used for experi- the following “low-energy” and “high-energy”
the pH was not readjusted after dilution of the ments shown in Fig. 3.) The lid was placed back protocols for microwave heating of 60 ml of
stock. on the steamer bowl, and the slides were buffer in a single Coplin jar. (Adjustment of
PBS. The composition of the PBS buffer steamed for 15 min. After the 15 min steaming, parameters may be required if two or more jars
used for immunohistochemistry and also in the rack was removed from the unit, and the are heated simultaneously.) To prevent boiling
some pretreatment experiments was 100 mM slides, with the tissue still covered by citrate of buffer, which could damage the tissue or dis-
sodium phosphate and 150 mM NaCl, pH 7.4. buffer, were allowed to cool at room tempera- lodge it from the slide, we used intermittent
Tris buffer. A 1.0 M Tris, pH 7.6 stock was ture for 2 min. Immunohistochemical process- rather than continuous heating.
prepared by adjusting the pH of Tris base with ing was then initiated beginning with a PBS Low-energy protocol. The buffer was pre-
HCl. Working Tris buffer solutions were pre- wash of the slides followed by application of heated in the microwave for 2 min 30 s at 30%
pared by diluting the 1.0 M stock with water. blocking solution as described below.
power, which warmed the buffer to ⬇96°C.
“Steamer/citrate” pretreatment of sections Alternative heating pretreatment methods Immediately after this preheating, the slides
before immunohistochemistry Glass staining dishes sometimes cracked when were immersed in the hot buffer. After 5 min
The steamer unit used was a food steamer (Os- heated in a water bath or in the microwave; (microwave off during this 5 min period), the
ter 6.1 quart; model 5712; available through thus, slides were incubated in plastic Coplin solution (with immersed slides) was reheated
retail and internet vendors). The “upper jars (catalog #25457-200; VWR International) for 30 s at 30% power. After another 5 min, the
steamer bowl” and “rice bowl” that came with for both the water bath and microwave proto- solution was again reheated for 30 s at 30%
the unit were not used. For slide incubation, a cols. These jars were filled with buffer (60 ml), power. After 16 min (5 min, 30 s, 5 min, 30 s,
polypropylene tube rack (catalog #66023-530; and for all equilibration and incubation peri- and 5 min), the Coplin jar was removed from
VWR International, West Chester, PA) was ods, the top was screwed onto the jar. The the microwave, and the solution (with slides
placed inside the lower steamer bowl; for incu- buffer temperature was measured immediately still immersed) was allowed to cool for 20 min
bation, slides were set on this rack. With this before and after slide incubation by stirring the at room temperature. Immunohistochemical
configuration, up to eight slides (25 ⫻ 75 mm) buffer with a thermometer. Before incubating processing was then initiated beginning with a
can be incubated at one time. A thermometer the slides in hot buffer, the slides were washed PBS wash of the slides and then blocking, as
was placed on the rack to allow continuous in PBS three times for 10 min. described below. During each 5 min cycle, the
monitoring of the temperature to which the Water bath. After equilibration of the buffer temperature declined from ⬇96°C at the be-
Tang et al. • Toolbox J. Neurosci., May 30, 2007 • 27(22):5837–5844 • 5839
ginning of the cycle to ⬇79°C at the end of the Immunohistochemical methods and images were assembled into photomon-
cycle. and imaging tages. Based on preliminary experiments, we
High-energy protocol. In this protocol, after Before immunohistochemical processing, an- adopted a longer exposure time for HCl-
preheating the buffer and immersing the slides, tigen retrieval was performed through pre- pretreated sections than for steamer/citrate-
the buffer was reheated much more frequently treatment using the protocols described in de- pretreated sections so that we could obtain
than in the “low-energy protocol.” The buffer tail above. Primary and secondary antibodies similar BrdU labeling intensity. The bound-
was preheated in the microwave for 2 min 30 s used are listed in Table 1. The sections were aries of the rostral migratory stream (RMS)
at 30% power, which warmed the buffer to washed with PBS three times for 10 min and were ascertained by examination of the Pax6
⬇96°C. Immediately after this preheating, the incubated for 1 h in blocking solution [2% v/v labeling. An area including part of the anterior
slides were immersed in the hot buffer and sub- normal goat serum (Vector Laboratories, Bur- portion of the subventricular zone (SVZa) and
jected to a continuous succession of heating lingame, CA) and 0.3% v/v Triton X-100 in vertical limb of the RMS was chosen for scor-
PBS]. The primary antibodies, diluted in ing. Each montage was masked, such that only
cycles in the microwave: 3 s on/1 s off at full
blocking solution, were applied, and the sec- a selected area was visible, and BrdU-labeled
power for 15 min (⬇225 cycles of 4 s). Using
tions were incubated with the primary anti- cells were scored by an observer blind to the
this protocol, the buffer did not boil, but the
bodies for 12–18 h at 4°C. For double labeling, pretreatment applied. The size of the counting
temperature was maintained at ⬇96°C antibodies were applied simultaneously. The area was determined using Canvas; this area
throughout the 15 min incubation period. The sections were next rinsed three times for 10 varied from 0.14 to 0.38 mm2 between animals
Coplin jar was then removed from the micro- min in PBS and subsequently incubated with but varied ⱕ15% for the adjacent steamer/
wave, and the solution (with slides still im- the appropriate secondary antibodies diluted citrate- and HCl-pretreated sections of a single
mersed) was allowed to cool for 20 min at in blocking solution for 1 h at room tempera- brain. From the count and area, the density of
room temperature. Immunohistochemical ture. To label all cell nuclei, the fluorescent nu- BrdU(⫹) cells was determined.
processing was subsequently initiated with a clear dye Hoechst 33342, DAPI, or Syto24 (Ta-
PBS wash of the slides and then blocking, as ble 1) was included with the secondary Results
described below. antibody solution. After a final rinse three After fixation, pretreatment of tissue be-
times for 10 min with PBS, the slides were cov- fore immunolabeling is required to visu-
HCl pretreatment erslipped with VectaShield (Vector Laborato- alize the commonly used proliferation
A glass Coplin jar was filled with 2.0 M HCl, the ries). Sections were examined using a Zeiss
marker BrdU. The standard pretreatment
cover was screwed on the jar, and the HCl was (Oberkochen, Germany) Axioscope fluores-
cence microscope equipped with a QImaging
is incubation of the tissue in dilute HCl,
equilibrated to 40°C in a water bath. Slides but this pretreatment impedes coimmu-
(Burnaby, British Columbia, Canada) Retiga
were then incubated in the 40°C HCl for 20 nolabeling for many other antigens and
1300 monochrome camera. The images shown
min. At the end of this incubation, the slides also blocks labeling by fluorescent DNA-
in the figures were collected using a 10⫻ Plan
were washed in 40 mM borate buffer two times
Neofluar lens [numerical aperture (NA), 0.30; binding dyes such as Hoechst 33342. We
for 15 min. This 40 mM borate buffer was pre-
Zeiss], except that for Figure 1 A, a 5⫻ Plan have developed a heat-induced epitope
pared by dissolving 7.6 g of sodium tetraborate Neofluar lens (NA, 0.15) was used. The cap- recovery method as an alternative to HCl
and 5.0 g of boric acid in 4 L of water and tured images were processed using Adobe Pho-
adjusting the pH to 8.5, which required ⬇100
pretreatment for BrdU immunolabeling
toshop (Adobe Systems, San Jose, CA) and and defined the conditions required for
l of 10.0 M NaOH/L buffer. After the borate Canvas (ACD Systems International, Victoria,
buffer wash, immunohistochemical processing reproducible high-quality labeling using
British Columbia, Canada). Within each fig-
was initiated with a PBS wash of the slides fol- ure, images showing labeling for the same an-
this method.
lowed by application of blocking solution, as tigen/target (for example, for BrdU), were all
described below. collected with the same exposure time and pro- Pretreatment of fixed sections using a
cessed identically. The images accurately repre- sodium citrate buffer and a steamer
BrdU injection and preparation of tissue sent the visual impression of observers. When allows high-quality labeling of BrdU
All animal procedures were approved by the tissue from an animal not injected with BrdU with concurrent labeling of other
Institutional Animal Care and Use Committee was labeled using the BrdU antibody, no nu- antigens and nuclear DNA
and the Emory Biosafety Committee of Emory clear BrdU signal was observed after either Incubating slides in a commercially avail-
University. “steamer/citrate” or HCl pretreatment (data able rice/vegetable steamer proved to be a
not shown).
Rat pups (postnatal day 1) were given an in- convenient way of exposing tissue to tem-
BrdU and Hoechst 33342 labeling for steam-
traperitoneal injection of BrdU (200 mg of
er/citrate versus HCl pretreatment were di-
peratures ⬃100°C. For experiments com-
BrdU per kilogram of body weight; stock is 10 paring the steamer/citrate method with
rectly compared in nine experiments. Relative
mg/ml BrdU in 0.007 M NaOH solution). labeling intensity with all other protocol per- HCl and other pretreatments, we intra-
Three hours later, the rats were anesthetized (5 mutations and for all other antibodies were di- peritoneally injected neonatal rats with
min in ice) and then perfused transcardially rectly compared with the standard steamer/ci- BrdU and then 3 h later, perfused these
with PBS (5 min at ⬇3 ml/min), followed by trate pretreatment in at least two to three rats with 4% paraformaldehyde fixative
cold 4% paraformaldehyde in PBS (5 min at experiments and cut 10 m parasagittal cryostat sec-
⬇3 ml/min). The brains were then removed
tions of the forebrain that included the
and postfixed overnight by immersion in 4%
Quantification of BrdU(⫹) cells SVZa and the RMS (Fig. 1 A). The neona-
paraformaldehyde in PBS at 4°C. After postfix- The detection sensitivity for cells incorporating
ation, the brains were cryoprotected by incuba-
tal SVZa and RMS are particularly suitable
BrdU was compared between steamer/citrate for these BrdU-labeling experiments be-
tion overnight in 30% w/v sucrose in PBS (un- and HCl-pretreated tissue as follows. For each
til they sank) and subsequently embedded in cause they have well defined borders
of the three brains scored, adjacent sections
OCT compound (catalog #25608-930; VWR (Luskin, 1993) and are comprised of a
with closely similar neuroanatomy were pre-
International), frozen with liquid nitrogen, treated by the steamer/citrate or HCl protocols highly proliferative neuronal progenitor
and sagittally sectioned at 10 m with a cryo- described above and then immunolabeled for cell population (Menezes et al., 1995; Pen-
stat. The sections were collected on SuperFrost BrdU and Pax6. Images were captured and cea and Luskin, 2003).
Plus slides (Fisher Scientific) and stored at processed as described above, except that a When BrdU labeling of tissue pre-
⫺20°C until used. 20⫻ Plan Neofluar lens (NA, 0.50) was used, treated by our steamer/citrate method was
5840 • J. Neurosci., May 30, 2007 • 27(22):5837–5844 Tang et al. • Toolbox
(Fig. 1G). However, compared with Table 2. Relative labeling intensity for antibodies as a function of pretreatment protocol
steamer/citrate-pretreated tissue, for the Quality of labeling
HCl-pretreated tissue, a much longer ex- Target (against) Steamer/citrate No pretreatment HCl Microwave (low)
posure was required to provide clear visu-
alization of the BrdU(⫹) cells for scoring. BrdU 4 – 2 –
PCNA 4 0 –1 0 –2 4
The quality of immunolabeling of tis- Phospho-H3 4 0 1–2 4
sue pretreated with the steamer/citrate Pax6 4 2–3 2–3 4
method was further evaluated by staining p27Kip1 4 0 0 –1 3
the sections with neuron-specific S100 4 3– 4 2–3 4
-tubulin antibody to assess the cytoar- Type III -tubulin 4 3 2–3 4
chitecture (supplemental Fig. 1, available DAPI 4 3– 4 – 4
Hoechst 33342 4 3 – 4
at www.jneurosci.org as supplemental
Syto 24 4 4 – 4
material) and whether neural cells known
4, Equal or almost equal to label intensity with citrate/steamer pretreatment; 3, distinctly weaker than citrate steamer, but still quite satisfactory; 2, quite
to be postmitotic, based on neuronal- weak relative to citrate/steamer, but still satisfactory; 1, very weak, but some specific label detectable; 0, no specific label observed (at 10⫻); –, no specific
specific nuclear protein (NeuN) labeling, label detected or marked reduction in labeled cell number, even when viewed at high power. Labeling intensity relative to steamer/citrate pretreatment was
were inappropriately also labeled by the subjectively scored by comparison of images collected using a 10⫻ Neofluar lens and an exposure time optimized for the steamer/citrate pretreatment slide
of the same experiment. Within each experiment, serial sections of a single brain were labeled; thus, little variability is expected between slides with respect
BrdU antibody (supplemental Fig. 2, to tissue quality and fixation. For each antibody, labeling intensity was scored for each pretreatment in at least two separate experiments. In every experi-
available at www.jneurosci.org as supple- ment, steamer/citrate was superior to HCl and no pretreatment, but there was some variability between experiments in relative labeling intensity for some
mental material). The cytoarchitecture of antibodies. When the scores varied, the range is indicated. In one experiment, the labeling intensity for Pax6 and S100 was slightly brighter for slides
pretreated with the microwave (low) protocol compared with the steamer/citrate protocol. In all other cases, the steamer/citrate protocol produced labeling
neurons was preserved at least as well in brighter than or equal to the microwave (low) protocol. PCNA, Proliferating cell nuclear antigen.
steamer/citrate-pretreated sections as in
HCl-pretreated sections, and although using the microwave (high) protocol (Fig. Buffer ionic strength is a
many NeuN(⫹) and many BrdU(⫹) cells 2C) or equilibrated to 97°C in a water bath critical parameter
were observed in sections immunostained (Fig. 2 E) resulted in BrdU labeling almost Pilot experiments led us to suspect that for
with both antibodies, no double(⫹) cells as intense as steamer citrate pretreatment heat pretreatment to reveal BrdU
were observed. (for details of treatment protocols, see epitopes, the ionic strength of the buffer
Materials and Methods). However, pre- in which the tissue is heated is a critical
Temperature is a critical parameter treatment by the microwave (low) proto- parameter determining labeling effective-
One previous study described successful col (Fig. 2 D) gave no specific BrdU label- ness. To test this, we compared a number
BrdU labeling by microwaving slides in ing, and pretreatment by incubation in of buffers to our standard 10 mM sodium
citrate buffer (Dover and Patel, 1994). 95°C citrate buffer (Fig. 2 F) yielded BrdU citrate, pH 6.0 buffer. Adding sucrose to
However, another group reported that labeling that was substantially less intense our standard buffer, which increases os-
this method was ineffective in their hands than that for the 99°C incubation [al- motic strength without changing ionic
(Valero et al., 2005). Before adopting the though still more intense than HCl (Fig. strength, had no effect on labeling inten-
steamer, we had performed heat-induced sity (Fig. 3, compare B, C with A). How-
2 B)]. Pretreatment by incubation in 90°C
epitope retrieval using a microwave oven ever, increasing the citrate concentration
buffer (Fig. 2G) yielded BrdU labeling that
to reveal p27Kip1 immunoreactivity (Li et (Fig. 3D) markedly diminished labeling,
was much less bright than for the 95°C
al., 2005). Although the microwave pre- not only for BrdU, but also for Pax6. Con-
pretreated tissue (Fig. 2 F). Collectively,
treatment protocol used for p27Kip1 (re- sistent with the results comparing differ-
these results indicate that for our tissue,
ferred to below as “low energy”) was inef- ent concentrations of citrate buffer, a di-
and using the citrate buffer, BrdU labeling
fective in revealing BrdU, we luted PBS solution (Fig. 3F ) gave good
serendipitously discovered that more in- intensity falls off sharply as the pretreat-
ment temperature decreases ⬍99°C. labeling, but the full strength stock (Fig.
tense microwave pretreatment (referred 3E) did not. Finally, 10 mM Tris buffer
to below as “high energy”) did reveal However, it is noteworthy that all of the
(Fig. 3G) gave excellent labeling, but 100
BrdU. We hypothesized that a critical pa- heating protocols (Fig. 2 A, C–G) gave ex-
mM Tris buffer (data not shown) did not.
rameter might be the intensity of heat cellent Pax6 and Hoechst labeling relative
This indicates that other buffers and pH
treatment and that the failure of Valero et to HCl pretreatment (Fig. 2 B). The results
values can be used successfully so long as
al. (2005) to reproduce the results of Do- of this experiment also suggest that the
the ionic concentration of the pretreat-
ver and Patel (1994) may have been attrib- efficacy of microwave pretreatment is en-
ment buffer solution is low. It is worth
utable to differences in the amount of tirely caused by the heating of the buffer
noting that pretreatment with 5 mM so-
heating of the buffer. and unrelated to other effects of the mi- dium citrate, pH 6.0, produced similar
To test this hypothesis, we compared crowave energy. Stated another way, vari- BrdU labeling intensity to 10 mM, but that
labeling of adjacent slides pretreated using ous methods that achieve a buffer temper- steamer treatment of tissue covered with 1
a number of different heating regimens ature of ⬃100°C will give similar, mM sodium citrate, pH 6.0, and water (0
(Fig. 2). For our steamer/citrate method, successful results. Finally, our results are mM) resulted in severe tissue damage.
we always preheat the steamer. With pre- consistent with the speculation that the
heating, we found the temperature within failure of Valero et al. (2005) to reproduce Effects on labeling quality of duration
the steamer unit (measured with a ther- the findings of Dover and Patel (1994) is of heating in steamer
mometer placed on the slide rack) to be attributable to Dover and Patel having Based on pilot experiments, we had settled
99°C throughout the 15 min slide incuba- achieved a buffer temperature of ⬇95°C on 15 min incubation in the steamer for
tion period. Relative to steamer citrate or greater but Valero et al. having only our “standard” steamer/citrate protocol.
pretreatment (Fig. 2 A), pretreatment of achieved a buffer temperature of ⬍90°C To determine the sensitivity of BrdU la-
slides immersed in citrate buffer heated with their microwave treatment. beling effectiveness to variations in
5842 • J. Neurosci., May 30, 2007 • 27(22):5837–5844 Tang et al. • Toolbox
Discussion
Major criteria for evaluating methods of
preparing tissue for BrdU immunohisto-
chemistry are (1) efficacy in revealing
BrdU labeling, (2) compatibility with co-
immunolabeling for other antigens, (3)
compatibility with pan-nuclear labels
such as Hoechst 33324, (4) the reproduc-
ibility of the procedure, and (5) the ease of Figure 2. Effective immunolabeling of BrdU using heat pretreatments requires a pretreatment temperature of ⬃100°C. A–G,
the procedure. In this study, we have char- ParasagittalcryostatsectionsofP1ratforebrainonglassslideswerepretreatedasindicatedattheleftofeachrow(fordetails,seeMaterials
acterized a steamer/citrate pretreatment and Methods) and then processed and imaged in parallel. Except for the HCl pretreatment (B), the pretreatment solution was 10 mM
protocol as an alternative to the HCl pre- sodium citrate, pH 6.0, and the duration of the pretreatment was 15 min. Compared with steamer pretreatment (A), pretreatment by
treatment method commonly used to re- immersion in buffer maintained at ⬃100°C by microwave heating (C) resulted in BrdU labeling of similar intensity, but a lower energy
veal BrdU. We find that steamer/citrate microwavepretreatment(D)producednospecificBrdUlabeling.Pretreatmentbyimmersioninbufferequilibratedto97°Cinawaterbath
pretreatment is superior to HCl for pre- (E), like high-intensity microwave treatment (C), yielded labeling almost as bright as steamer pretreatment. When sections were pre-
treatedbyimmersionin95°Cbuffer(F),BrdUlabelingintensitywasmuchreducedcomparedwithsteamerpretreatment,andimmersion
treatment of paraformaldehyde-fixed sec-
inbufferat90°Cwasevenweaker(G).Forsectionspretreatedbyincubationinbufferat85°C,nospecificBrdUlabelingwasdetected,even
tions with respect to each of these five cri- withlongexposuretimes(datanotshown).Allpretreatments,exceptHCl(B),producedstrongPax6andHoechstlabeling.Exposuretimes
teria. Furthermore, compared with HCl inthisfigure(shownatthetopofeachcolumn)wereoptimizedforthesteamerpretreatment.Withtheseexposuretimes,BrdUandPax6
pretreatment, steamer/citrate may reduce labeling are barely detectable for the slide pretreated with HCl. For the HCl pretreatment, no labeling with Hoechst was detectable even
costs by allowing use of smaller amounts withlongexposuretimes.Ineachimage,thesolidlinesdemarcatetheborderofthelateralventricle,anddashedlinesoutlinetheboundary
of expensive BrdU antibodies, as well as between the SVZa and adjacent neural tissue. Within each row, the solid and dashed lines are identically positioned in each image. Scale
other antibodies. bar: G (for A–G), 200 m.
Tang et al. • Toolbox J. Neurosci., May 30, 2007 • 27(22):5837–5844 • 5843
the steamer is more convenient and pro- ized device to attain buffer temperatures P19INK4D null mice. Soc Neurosci Abstr
duces slightly superior results. sufficient for optimal labeling. 31:143.23.
Luskin MB (1993) Restricted proliferation and
Although Dover and Patel (1994) and In summary, use of the steamer/citrate
migration of postnatally generated neurons
we achieved good results using microwave protocol reported here is expected to facil- derived from the forebrain subventricular
heat treatment of tissue, Valero et al. itate the investigation of characteristics of zone. Neuron 11:173–189.
(2005), in a study designed to identify proliferating cell populations by allowing Menezes JR, Luskin MB (1994) Expression of
“optimized” methods for BrdU and colabeling with a wider range of antibod- neuron-specific tubulin defines a novel popu-
PCNA (proliferating cell nuclear antigen) ies than is possible with HCl pretreatment lation in the proliferative layers of the devel-
labeling in the rostral migratory stream of and simplifying determining total cell oping telencephalon. J Neurosci 14:
rodents, the same region that we studied, number in regions scored for BrdU- 5399 –5416.
Menezes JR, Smith CM, Nelson KC, Luskin MB
reported that the protocol of Dover and labeled cells. The procedure we have de-
(1995) The division of neuronal progenitor
Patel (1994) “proved to be ineffective.” veloped will be useful in many applica- cells during migration in the neonatal mam-
There are two likely reasons for the lack of tions. For example, nuclear labeling in malian forebrain. Mol Cell Neurosci
success Valero et al. had with hot citrate conjunction with BrdU labeling will allow 6:496 –508.
pretreatment: (1) the buffer they used was a more precise determination (when us- Miller MW, Nowakowski RS (1988) Use of
100 mM sodium citrate, which we found ing z-stacks) of whether a cell is a neuron bromodeoxyuridine-immunohistochemistry
to yield much weaker staining than 10 mM or satellite glial cell in contentious cases. to examine the proliferation, migration and
sodium citrate (Fig. 3, compare A, D), and More generally, the steamer/citrate time of origin of cells in the central nervous
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(2) Dover and Patel (1994) make no men- method may promote our understanding
Nowakowski RS, Hayes NL (2000) New neu-
tion of the buffer temperature as a func- of neurodevelopment and lead to ad- rons: extraordinary evidence or extraordinary
tion of time for their microwave treat- vances in the identification of mitotically conclusion? Science 288:771.
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