Step-by-Step In Situ Hybridization Method for Localizing
Gene Expression Changes in the Brain
Jorge J. Palop, Erik D. Roberson, and Inma Cobos
RNA in situ hybridization is a powerful technique for examining gene expression in specific cell populations.
This method is particularly useful in the central nervous system with its high cellular diversity and dynamic
gene expression regulation associated with development, plasticity, neuronal activity, aging, and disease.
Standard quantitative techniques such as Western blotting and real-time PCR allow the detection of
altered gene or protein expression but provide no information about their cellular source or possible
alterations in expression patterns. Here, we describe a step-by-step RNA in situ hybridization method
on adult and embryonic brain sections for quantitative neuroscience. We include fully detailed protocols
for RNase-free material preparation, perfusion, fixation, sectioning, selection of expressed sequence tag
cDNA clones, linearization of cDNA, synthesis of digoxigenin-labeled RNA probes (riboprobes), in situ
hybridization on floating and mounted sections, nonradioactive immunohistochemical detection of ribo-
probes for light and fluorescence microscopy, and double labeling. We also include useful information
about quality-control steps, key online sites, commercially available products, stock solutions, and storage.
Finally, we provide examples of the utility of this approach in understanding the neuropathogenesis of
Alzheimer’s disease. With virtually all genomic coding sequences cloned or being cloned into cDNA
plasmids, this technique has become highly accessible to explore gene expression profiles at the cellular
and brain region level.
Key words: In situ hybridization, RNA, RNA probes, Riboprobes, Brain sections, Sliding microtome,
Cryostat, EST, Expressed sequence tag, cDNA, In vitro transcription, Transcripts, Digoxigenin-
labeled probes, NTB, BCIP, HNPP, Fast red, Light and fluorescence microscopy, Alzheimer’s disease,
Frontotemporal dementia, NPY, ARC, Nav1.1
The central nervous system is an extremely complex structure
containing hundreds of distinct cell types. This diversity is also
present at the local level. Circuits or local networks are usually
assembled through contributions from a diverse array of cells with
Erik D. Roberson (ed.), Alzheimer’s Disease and Frontotemporal Dementia, Methods in Molecular Biology, vol. 670,
DOI 10.1007/978-1-60761-744-0_15, © Springer Science+Business Media, LLC 2011
208 Palop, Roberson, and Cobos
distinct gene expression profiles. In addition, patterns of gene
expression often overlap between cell types and are dynamically
regulated by age, synaptic plasticity, brain activity, aging, or disease.
This cellular diversity and the dynamic regulation of gene expression
pose tremendous challenges for understanding in vivo regulation
of gene expression. Standard quantitative techniques such as
Western blotting and real-time PCR can detect changes in gene
or protein expression, but provide no information about the cellular
source. This missing piece of information makes it very difficult to
assess the functional significance of a given change in expression
level. For example, a change could have opposite functional out-
comes depending on whether it occurred in excitatory or inhibi-
tory neurons. In situ hybridization is a powerful technique that
effectively addresses cellular localization of gene expression.
Two landmark projects, the Integrated Molecular Analysis of
Genomes and their Expression (IMAGE) Consortium and the
National Institutes of Health’s Mammalian Gene Collection
(MGC), have made virtually all human and mouse genomic cod-
ing sequences accessible in cDNA plasmids, which can be readily
used to generate RNA probes for in situ hybridization. Although
there are remarkable efforts to map expression patterns of thou-
sands of genes in normal brain tissue (for example, the Allen
Institute for Brain Science at http://www.brain-map.org/;
Mousebrain Gene Expression Map at St. Jude Children’s
Research Hospital at http://www.stjudebgem.org/; GenePaint.
org at http://www.genepaint.org/), these sources are not
designed to address alterations in gene expression patterns
induced by experimental manipulations or disease processes,
which can be critical for understanding pathogenic processes.
For example, we found de novo, ectopic neuropeptide Y (NPY)
gene expression in the granule cells of transgenic mouse models
of Alzheimer’s disease (Fig. 2d). The NPY gene, which is exclu-
sively expressed by GABAergic inhibitory cells in healthy brain,
is transcribed ectopically in glutamatergic cells in the disease
state, with important implications for the function of the hip-
pocampal circuit (1). Although granule cells are easily identifi-
able by anatomy, in other cases, additional information is
necessary to identify specific cellular sources. For example, the
sodium channel Nav1.1 is expressed in cells of the CA1 hip-
pocampal region (Fig. 1d, left). However, only after double
labeling for Nav1.1 mRNA and parvalbumin it is possible to
identify the specific cellular source of Nav1.1 as parvalbumin-
positive inhibitory interneurons, rather than pyramidal cells
(Fig. 1d, right).
In situ hybridization is the procedure for labeling a particular
sequence of DNA or RNA with a specific complementary detec-
tion probe. Originally described by Gall and Pardue (2) and
John et al. (3), this method has been extensively used and adapted
209In Situ Hybridization Method for Localizing Gene Expression Changes
Fig. 1. Overview of the in situ hybridization method. (a) mRNA is transcribed from the DNA template (anti-sense strand, light
blue) by RNA polymerases moving from 3¢ to 5¢ along the template, as indicated by the arrow. The mRNA or transcript is
synthesized 5¢ to 3¢ and contains a 5¢UTR (untranslated region, yellow ), a coding sequence (green), a 3¢UTR (blue), and a
poly-A signaling tail (red ). Transcripts or expressed sequence tags (ESTs) from virtually all mouse genes have been cloned into
cDNA plasmids, which can be readily used to generate RNA in situ probes. RNA probes can target the full length of the tran-
script, including coding sequences and untranslated regions, or only parts. RNA probes are synthesized in vitro from the cDNA
coding or sense strand of EST clones by RNA polymerases. This synthesis generates a digoxigenin-labeled RNA probe comple-
mentary to the mRNA. (b) RNA in situ hybridization is the procedure by which a particular mRNA is hybridized with a specific,
complementary, labeled RNA probe. The process includes the hybridization of an mRNA with the digoxigenin-labeled complementary
riboprobe, immunohistochemical detection of digoxigenin, and development for light or fluorescence microscopy. (c) Activity-regulated
cytoskeletal-associated protein (Arc) in situ hybridization for light microscopy on 30-mm floating sections. Note intense labeling
in some, but not all, granule cells in the dentate gyrus of the hippocampus (for more info, see (4)). (d) Double in situ hybridization
for Nav1.1 (red) and immunohistochemistry for parvalbumin (PV, green) for fluorescence microscopy on 10-mm cryosections.
Note that Nav1.1 is expressed in PV-positive GABAergic interneurons.
210 Palop, Roberson, and Cobos
Fig. 2. Synthesis of RNA probes from cDNA clones. The IMAGE and MGC consortiums have created extensive cDNA librar-
ies of ESTs and made them available to the research community. (a) There are approximately 40 cDNA clones available
for mouse neuropeptide Y (NPY). The diagram illustrates one of them (IMAGE: 5683102), containing the NPY full-length
transcript inserted into a pYX-Asc vector. It is important to know the polymerase promoters (in this case, T3 and T7), the
restriction enzyme sites flanking the fragment (NotI and EcoRI), and the direction of the transcript (3¢ at NotI and 5¢ at
EcoRI). (b) Sense and anti-sense RNA probes are generated as illustrated. NPY anti-sense riboprobe, which will hybridize
211 In Situ Hybridization Method for Localizing Gene Expression Changes
for studying gene expression in a wide variety of animal models,
tissues, and experimental conditions. This paper focuses on the
detection of messenger RNA (mRNA) on brain tissue sections by
complementary RNA probes (riboprobes). We have been opti-
mizing this technique over the course of several years of studying
adult and embryonic brain tissue from mouse models of
Alzheimer’s disease and neurodevelopmental disorders (1, 4–6).
The protocols described are suitable for adult or embryonic tis-
sue, floating or mounted sections, and a broad range of mRNA
lengths (200–3,000 bp). We describe step-by-step and fully
detailed protocols for RNase-free material preparation, perfusion,
fixation, sectioning, selection of cDNA clones, synthesis of
digoxigenin-labeled riboprobes, in situ hybridization on floating
and mounted brain sections, immunohistochemical detection of
riboprobes for light and fluorescence microscopy, and double in
situ and immuno detection. We also include useful information
about tips, quality-control steps, key online sites, commercially
available products, and storage.
2. RNase-inactivating wipes.
3. Autoclave and oven.
4. Aluminum foil.
5. RNase-free water (see Note 1).
6. 0.22-mm Filter units.
All of the reagents listed below must be prepared using RNase-
free conditions, as described in Subheading 3.1.
1. Anesthetic agent, such as pentobarbital.
2. Perfusion pump or large-capacity syringes and tubing.
3. Surgical tools.
2.1. Avoiding RNase
Fig. 2. (continued) with NPY mRNA in tissue sections, can be synthesized after linearizing the plasmid with EcoRI and
transcribing it with T3 RNA polymerase. The sense riboprobe, which does not hybridize with NPY mRNA, serves as a
control and can be synthesized after linearizing the plasmid with NotI and transcribing it with T7 RNA polymerase.
(c) cDNA clones, DNA templates, and riboprobes are run in agarose gels to verify size and quality. Lanes 1–3 and 4–6
represent two different cDNA clones. (d) NPY in situ hybridization for light microscopy on 30-mm floating sections from a
normal (non-transgenic; upper panel) and an Alzheimer’s disease (AD) mouse (for more info, see (1)). Note the ectopic
expression of NPY in the granule cells of the hippocampus in the AD mouse.
212 Palop, Roberson, and Cobos
4. Normal saline: 0.9% NaCl.
5. 2× Phosphate buffer (2× PB): 160 mM Na2HPO4, 400 mM
NaH2PO4, pH 7.4. For a 1-L stock solution, dissolve 22.72 g
of Na2HPO4 and 5.52 g of NaH2PO4 × 1H2O in diethyl pyro-
carbonate (DEPC)-treated water, and adjust pH to 7.4. Filter
through 0.22-mm filter. Store at room temperature for weeks
or at −20°C for months.
6. 1 M NaOH: 40 g of NaOH in 1 L of water.
7. 1× Phosphate buffer (1× PB): Prepared from 2× PB by dilut-
ing 1:1 in DEPC-treated water.
8. 8% Paraformaldehyde (8% PFA, w/v): For 1 L, measure 80 g
of PFA (see Note 2). Add about 600 ml of DEPC-treated
water that has been heated to ~65°C in a microwave. Place
the mixture on a hotplate in a fume hood and stir with
continued heating until the solution reaches, but does not
exceed, 85°C (see Note 3). Turn off the heat and continue
stirring. At this point, the solution should be semi-white
and cloudy. Add 2–4 drops of 1 M NaOH and wait for
2–4 min. The solution will become less cloudy and more
transparent. Repeat the addition of NaOH until the PFA
goes fully into solution. The goal is to obtain a completely
transparent solution with minimal addition of NaOH,
because high concentrations of NaOH increase the pH and
affect the fixative properties of PFA. Add heated water to a
volume of 1 L. Let the solution cool to room temperature
while stirring. For transcardial perfusion, filter the solution
with a 0.22-mm pore size. Aliquot and store at 4°C for use
within 1–2 days or at −20°C for any longer period of
9. 4% PFA in 1× PB: Prepared as a 1:1 mixture of 8% PFA and
2× PB. If the solutions are stored at −20°C, thaw in a 40–50°C
water bath. Wait until both solutions are transparent. Do not
use a microwave to thaw PFA.
10. Phosphate-buffered saline (PBS): Purchase 10× sterile stock
solution (pH 7.4), dilute to 1×.
11. 30% Sucrose in PBS (w/v).
12. Sliding microtome, cryostat, or vibratome.
13. 10% Sucrose in PBS.
14. Cryoprotectant solution (for microtome and vibratome sec-
tions): 30% ethylene glycol, 30% glycerin, 40% 1× PBS
(v/v/v). Store at −20°C.
15. Embedding molds (for cryostat sections).
16. TBS medium (Triangle Biomedical Science, for cryostat
sections of postnatal and adult brains) or Tissue-Tek OCT
(for cryostat sections of embryonic brains).
17. Superfrost Plus slides (Fisher Scientific).
213In Situ Hybridization Method for Localizing Gene Expression Changes
1. EST clones for the gene of interest (see Subheading 3.3.1
below for instructions on selection).
2. T7, T3, and/or SP6 sequencing primers (see Subheading 3.3.2
below for instructions on selection). Standard primers for
each are available from a variety of commercial sources.
3. Restriction enzymes and buffers (see Subheading 3.3.2 below
for instructions on selection).
6. UltraPure phenol:chloroform:isoamyl alcohol solution (25:24:1,
Invitrogen). Store in 1 ml aliquots at 4°C or −20°C.
7. 3 M Sodium acetate buffer solution, pH 7.0 (Sigma).
8. 100% Ethanol.
9. 70% Ethanol.
10. TE buffer: 10 mM Tris–HCl, pH 8.0, 1 mM EDTA, in
13. Agarose gel electrophoresis apparatus and power supply.
15. Tris–acetate EDTA (TAE) buffer: 40 mM Tris–acetate, 1 mM
EDTA. To prepare a 50× TAE stock solution, dissolve 242 g
of Tris base in approximately 750 ml of deionized water. Add
57.1 ml of glacial acid and 100 ml of 0.5 M EDTA (pH 8.0),
and adjust the solution to a final volume of 1 L. The pH of
this buffer should be about 8.5 and does not need to be
adjusted. Store at room temperature.
16. Ethidium bromide solution, 10 mg/ml (Bio-Rad).
17. BlueJuice buffer (Invitrogen).
18. 1-kb DNA ladder.
19. T7, T3, and/or Sp6 RNA polymerase (Promega). Store at
20. 5× Transcription buffer (Promega, provided with polymerase
enzyme). Store at −20°C.
21. 100 mM dithiothreitol (DTT; Promega, provided with poly-
merase enzyme). Store at −20°C.
22. 10× Digoxigenin-labeled nucleotide mix (Roche DIG RNA
labeling mix: contains ATP, CTP, GTP, UTP, and DIG-11-
UTP). Store at −20°C.
23. Protector RNase inhibitor (Roche). Store at −20°C.
24. RNeasy Mini Kit for RNA purification (Qiagen).
25. 100% UltraPure formamide, redistilled (Invitrogen). Store
214Palop, Roberson, and Cobos
1. 24-Well plates (for floating sections).
2. Clean glass Pasteur pipettes with their tips sealed in a flame,
for handling sections.
3. PBS/0.5% Tween: 0.5% Tween-20 in 1× PBS, pH 7.4.
4. Shaking platform.
5. 4% PFA in PBS: Dilute 8% PFA stock in 2× PBS.
6. Tris/EDTA/Tween buffer: 50 mM Tris–HCl, pH 8.0, 5 mM
EDTA, 0.5% Tween-20. For 100 ml, mix 94 ml of DEPC-treated
water, 5 ml of 1 M Tris–HCl, pH 8.0 stock solution, 1 ml of
500 mM EDTA stock solution, and 500 ml of 100% Tween-20.
7. Proteinase K buffer: 50 mM Tris–HCl, pH 8.0, 5 mM EDTA,
0.5% Tween-20, 1 mg/ml recombinant, PCR-grade proteinase
K (Roche 3 115 828). Add 1 ml of Proteinase K stock solu-
tion (~20 mg/ml) per 20 ml of Tris/EDTA/Tween buffer.
Omit Tween-20 for cryostat sections.
8. Acetylation solution: 1.355% triethanolamine, 0.175% HCl,
and 0.25% acetic anhydride. Mix 9.8 ml of water, 133.5 ml of
triethanolamine (Fisher Chemical, certified), and 17.5 ml
of 12 M HCl in a safety hood. Once dissolved, add 25 ml of
acetic anhydride (Sigma, reagent grade, ³98%), mix well, and
9. 50× Stock Denhardt’s solution: 1% bovine serum albumin
(BSA), 1% Ficoll, 1% polyvinyl pyrolidone. To prepare 100 ml,
mix 1 g of BSA, 1 g of Ficoll PM 400 (Type 400, Sigma), 1 g
of polyvinyl pyrolidone, average molecular weight 40,000
(Sigma), and 100 ml of water. Warm mixture to dissolve each,
filter, and aliquot to 15 ml each. Store at −20°C. 50×
Denhardt’s solution is also commercially available.
10. In situ pre-hybridization buffer: 50% formamide, 5× salt
sodium citrate (SSC) buffer, pH 7.0, 5× Denhardt’s solution,
0.25 mg/ml salmon sperm DNA, 0.5 mg/ml yeast tRNA.
To prepare a 200-ml stock solution, mix 100 ml of 100%
UltraPure formamide (Invitrogen), 50 ml of 20× SSC
(Roche), 20 ml of 50× Denhardt’s, 30 ml of water, 50 mg of
UltraPure salmon sperm DNA (Invitrogen), and 100 mg of
yeast tRNA (Invitrogen). Store at −20°C.
11. Slide humidity incubation box.
12. Precise temperature control oven (see Note 4).
13. 5× SSC/0.5% Tween buffer: Prepare from commercially pur-
chased 20× SSC (Roche) and Tween-20. For cryostat sections,
use 5× SSC without Tween.
14. 0.2× SSC/0.5% Tween buffer: Prepare from commercially
purchased 20× SSC (Roche) and Tween-20. For cryostat
sections, use 0.2× SSC without Tween.
2.4. RNA In Situ
215In Situ Hybridization Method for Localizing Gene Expression Changes
15. Tris-HCl/saline/Tween buffer: 100 mM Tris–HCl, pH 7.5,
150 mM NaCl, 0.5% Tween-20. To prepare 1 L, mix 870 ml
of water, 100 ml of 1 M Tris, pH 7.5, 30 ml of 5 M NaCl
stock, and 5 ml of Tween. For cryostat sections, omit the
16. Heat-inactivated sheep serum (HISS): Heat sheep serum
(Sigma) at 56°C for 20 min.
17. Blocking buffer: 10% HISS in Tris-HCl/saline/Tween
buffer. For cryostat sections, omit the Tween-20.
18. Alkaline phosphatase-conjugated anti-digoxigenin antibody
(Anti-digoxigenin-AP, Fab fragment, from sheep; Roche).
19. PBS/EDTA solution: 0.1 M PBS, 1 mM EDTA pH 8.0, 0.5%
Tween. For 1,000 ml of total volume, mix 900 ml of water,
100 ml of 10× PBS, 2 ml of 0.5 M EDTA, and 2.5 ml of
Tween 20. Omit the Tween for cryostat section.
20. 1% Gelatin (for mounting floating sections). Mix 1 g of gela-
tin from porcine skin, type A (Sigma) in 100 ml of warm
22. RNase-free glass coverslips.
For light microscopic detection:
23. NTMT Buffer: 0.1 M Tris–HCl, pH 9.5, 0.1 M NaCl, 50 mM
MgCl2. To prepare 400 ml, mix 332 ml of water, 40 ml of 1 M
Tris, pH 9.5, 8 ml of 5 M NaCl, and 20 ml of 1 M MgCl2.
24. NBT/BCIP stock solution: 18.75 mg/ml nitroblue tetrazolium
chloride and 9.4 mg/ml 5-bromo-4-chloro-3-indolyl-phosphate,
toluidine salt in 67% dimethyl sulfoxide (DMSO; Roche).
25. #1 Size sable paintbrush.
26. Cytoseal or Permount mounting medium.
For fluorescent microscopic detection:
27. Fluorescence detection buffer: 100 mM Tris–HCl, pH 8.0,
100 mM NaCl, 5 mM MgCl2. To prepare 1 L, mix 100 ml of
1 M Tris–HCl, pH 8.0, 20 ml of 5 M NaCl, and 5 ml of 1 M
28. HNPP/Fast Red TR developing mix: 100 mg/ml HNPP,
250 mg/ml Fast Red TR, 100 mM Tris–HCl pH 8.0, 100 mM
NaCl, and 5 mM MgCl2. To prepare 1 ml, mix 10 ml of
10 mg/ml HNPP in dimethylformamide and 10 ml of 25 mg/
ml Fast Red in redistilled H2O with 1 ml of fluorescence
detection buffer. We purchase HNPP and Fast Red TR
together in the HNPP fluorescence detection set (Roche).
29. Fluorescence mounting medium.
216Palop, Roberson, and Cobos
The first step of in situ hybridization is to create a clean environment
to preserve the RNA from the samples. mRNA is very susceptible
to degradation by ribonucleases (RNases). To ensure maximal
mRNA preservation, all solid and liquid materials must be free of
RNases and stored in semi-sterile condition throughout the
1. Assign an RNase-free dedicated area at your bench to keep all
RNase-free solutions and materials. Regularly wipe your work
area and equipment with RNase-free wipes.
2. Avoid skin contact with reagents and work surfaces, and use
clean gloves at all times.
3. All glassware (e.g., Coplin jars, beakers, cylinders, and cover-
slips) and metal tools (i.e., forceps, scissors, and stir bars) used
during the perfusion, sectioning, and staining must be baked.
Seal the glassware opening, or wrap up the metal materials
with aluminum foil and bake for 6 h at 220°C in an oven.
Sterile plastic products are considered RNase-free material
and do not need to be autoclaved.
4. Prepare RNase-free water. All buffers used during the proce-
dure must be prepared using water treated with 0.1% diethyl
pyrocarbonate (DEPC, Sigma D-5758), a standard method
to inactivate RNases (see Note 5). Mix 1 ml of DEPC per
liter of milli-Q water, and stir for 10 h at room temperature.
Autoclave 0.1% DEPC-treated water for 30 min at 125°C to
eliminate residual DEPC. Store for weeks at room tempera-
ture. We routinely prepare batches of 5 L at a time.
5. Ensure that all buffers for subsequent steps (including perfu-
sion and fixation) are RNase free by purchasing RNase-free
reagents, using autoclaved glassware and tools, preparing the
solutions with DEPC-treated water, and filtering through a
0.22-mm pore size membrane. Solutions that do not have pri-
mary amine groups can be treated directly with DEPC and
then autoclaved (but see Note 6 regarding Tris-based solu-
tions). Keep the bottles in an RNase-free designated location
and make sure that these products are always handled with
clean gloves (see Notes 7 and 8).
1. If a perfusion pump is used, clean the tubing by flushing
first with 1 M NaOH for 15–30 s and then with DEPC-
treated water. Sterile syringes and needles can be used as
2. Anesthetize animals.
3.1. Avoiding RNase
3.2. Perfusing, Fixing,
and Sectioning Brain
3.2.1. Perfusing and Fixing
217In Situ Hybridization Method for Localizing Gene Expression Changes
3. Flush-perfuse transcardially with 1× PB or normal saline for
10–20 s until most of the blood is washed out from the circu-
4. Perfuse with 4% phosphate-buffered PFA (4% PFA in 1× PB)
for 5 min (see Notes 9 and 10). Keep the 4% PFA at 4°C or
on ice during the perfusion.
5. Remove the brain, cut into hemibrains, and drop-fix in the
same fixative (4% PFA in 1× PB) for 24–48 h at 4°C. Make
sure that the fixative volume is at least ten times larger than
the volume of the brain sample.
6. After the brains have been fixed at 4°C for 24–48 h, rinse
twice for 2–5 h with PBS at 4°C. This reduces background
7. For immunohistochemistry, brains can be stored for 2–4 weeks
at 4°C before sectioning. For in situ hybridization, brains
must be sectioned immediately to reduce RNA degradation.
We recommend either microtome (Subheading 3.2.2) or cry-
ostat (Subheading 3.2.3) sectioning. A vibratome can also be
used, although it provides less accurate sectioning through-
out the extent of the sample.
1. Transfer the brains into 30% sucrose in PBS for 24 h at 4°C.
Initially, the brains will float. They will sink when cryopro-
tected and ready for sectioning; do not section until the brains
sink. Make sure that the volume of sucrose solution is at least
ten times greater than the volume of the sample.
2. Place the hemibrains on the freezing stage of the microtome
at room temperature and add a few drops of 10% sucrose
around the hemibrains (see Note 11). Once the stage tem-
perature is lowered, the 10% sucrose base will freeze and
firmly attach the samples to the stage.
3. Set the temperature of the freezing stage at −20°C and wait
for 5–10 min until the hemibrain and 10% sucrose base are
4. Collect ten subseries of floating sections (30 mm) per mouse
hemibrain in 1.5-ml Eppendorf tubes with 1.2 ml of ethylene
glycol-based cryoprotectant medium. Each tube will contain
8–12 equidistant sections, 300 mm apart, throughout the
rostro–caudal extent of the hemibrain. (These are standard
parameters that could change depending on the experiment
or animal model.)
5. Store the sections at −20°C until use.
1. Transfer the brains into 30% sucrose in PBS for 24 h at 4°C.
Initially, the brains will float. They will sink when cryoprotected
and ready for sectioning; do not section until the brains sink.
3.2.3. Cryostat Sectioning
218Palop, Roberson, and Cobos
Make sure that the volume of sucrose solution is at least ten
times greater than the volume of the sample.
2. Embed the hemibrains in tissue-freezing medium using embed-
ding molds. For better embedding, gently remove excess 30%
sucrose from the surface of the brains before embedding. For
postnatal and adult brain tissue, use TBS medium. For embry-
onic brains, use Tissue-Tek OCT compound.
3. For TBS medium, allow the tissue to set for 3–5 min at room
temperature while gently stirring the brain with a sterile
pipette tip. For OCT medium, allow the tissue to set for 1 h
at 4°C on a shaker or stir regularly.
4. After orienting the sample, freeze on dry ice and wait for
30–60 min before cutting the samples in a cryostat or storing
them at −80°C.
5. Set the cryostat at −25°C. Note that different embedding
media have different optimal temperatures (around −25°C
for TBS and −18°C for OCT).
6. Collect 20 subseries of cryosections (10 mm) per mouse hemi-
brain on RNase-free Superfrost Plus slides. Each slide will
contain 6–8 equidistant sections, 200 mm apart, throughout
the rostro–caudal extent of the hemibrain. These are standard
conditions that might change depending on the experiment.
7. Store the slides at −80°C in a clean slide box with desiccant
caps. Place each slide box inside of a plastic zip bag.
Anti-sense and sense RNA probes (riboprobes) are generated
from a linearized cDNA plasmid containing the complete or a
partial transcript of a particular gene. The mRNA transcript of a
gene includes the 5¢UTR (untranslated region), the coding
sequence, the 3¢UTR, and a poly-A tail (Fig. 1a). RNA probes
can target any sequence of the transcript. In 1993, the Integrated
Molecular Analysis of Genomes and their Expression (IMAGE)
Consortium was created with the primary goal of creating exten-
sive cDNA libraries and associated bioinformatics tools, and mak-
ing them available to the research community (more information
at http:/ /image.hudsonalpha.org/). In addition, the Mammalian
Gene Collection (MGC), a trans-NIH initiative, provides research-
ers with sequence-validated full-length protein-coding cDNA
clones (http:/ /mgc.nci.nih.gov/). These cDNA plasmids or clones
called expressed sequence tags (ESTs) are commercially available
through IMAGE, Invitrogen, American Type Culture Collection
(ATCC), Open Biosystems, RIKEN, and other sources. Virtually
all human and mouse gene transcripts or expressed sequences
have been cloned into EST clones. This creates a tremendous
resource of expressed sequences ready to be used for in situ
3.3.1. Selecting cDNA/EST
219In Situ Hybridization Method for Localizing Gene Expression Changes
1. The first step is to identify two or three EST clones per gene
of interest by using an online genome browser (e.g., http:/ /
www.ncbi.nlm.nih.gov/unigene or http:/ /genome.ucsc.edu).
Search for the name of the gene and organism to find the
gene’s web page. mRNA and EST sequences are constantly
updated in these web pages. Select cDNA clones containing
full or partial transcript sequences of lengths of 200–3,000 bp
(ideally 500–1,000 bp). Although an RNA probe can be
selected from the 3¢UTR, 5¢UTR, or the coding region of a
gene, the 3¢UTR regions usually reduce the probability of
cross-hybridization with other genes. Throughout this chap-
ter, we will use in situ hybridization for mouse NPY as an
example. Mouse NPY has six mRNA and 40 EST sequences.
One of them, an IMAGE cDNA clone (IMAGE:5683102;
MGC:57879), contains the full transcript sequence of NPY
(561 bp), including 77 bp of 5¢UTR, 294 bp of coding
sequence, 176 bp of 3¢UTR, and 18 bp of poly-A tail (Fig. 2a).
We successfully used this clone in a recent publication (1).
2. Once two or three EST clones per gene have been selected, it
is important to determine whether the sequence of the
selected clone is specific for the gene of interest by perform-
ing a BLAST search against the full genomic transcript
(http:/ /www.ncbi.nlm.nih.gov/BLAST/). Avoid clones with
sequences that overlap with other genes. Using the 3¢UTR
regions reduces the probability of cross-hybridization with
3. In an EST clone, a transcribed sequence is inserted into a
cloning vector. Check whether the selected cDNA plasmid
contains RNA polymerase promoter sites (i.e., T7, T3, and
SP6) and determine the orientation of the transcript sequence.
The query tool at http:/ /image.hudsonalpha.org/html/
query_tools.shtml or the web sites of EST clone providers are
useful for this purpose. For example, our NPY complete tran-
script sequence cDNA (IMAGE:5683102) was cloned into
the pYX-Asc vector with the 5¢ end at EcoRI and 3¢ end at
NotI (Fig. 2a). This information is used later to generate the
4. Once cDNA clones are received, sequence them using prim-
ers based in the polymerase promoters to verify the gene and
the orientation of the coding sequence (Fig. 2b).
5. Perform a BLAST search for the resulting sequences and con-
firm that they correspond with the gene of interest.
6. Check the sequence orientation and determine which pro-
moter will synthesize the sense and anti-sense strands (Fig. 2).
Because the sequencing primers are complementary to the
polymerase promoter, the sequence generated represents
220Palop, Roberson, and Cobos
what the polymerase will transcribe. If the sequencing reaction
from a given polymerase promoter generates a sequence
complementary to the mRNA (termed plus/minus), then the
polymerase will transcribe an “anti-sense” RNA probe, which
will be used to detect the mRNA of interest during the in situ
hybridization. If the sequencing reaction from a primer com-
plementary to the polymerase promoter generates a sequence
equivalent to the mRNA (termed plus/plus), then the pro-
moter will transcribe a “sense” RNA probe, which will be
used as a control. In Fig. 2b, the sequencing primers that
bind to T3 would produce a sequence complementary to the
mRNA (plus/minus), so T3 polymerase is used to synthesize
the anti-sense riboprobe. Sequencing primers that bind to
T7 would produce a sequence equivalent to the mRNA
(plus/plus), so T7 polymerase is used to generate the sense
7. Once the cDNA clones containing the full or partial tran-
script sequence of the gene of interest have been sequenced
and verified, it is useful to elaborate EST clone maps contain-
ing information about the location of polymerase promoters
and restriction enzymes, as well as the direction of the tran-
script sequence and sizes (Fig. 2a).
To generate riboprobes, cDNA plasmids are first linearized with
vector-specific restriction enzymes (steps 1–4). Then the linear-
ized template is purified from the restriction enzyme solution
(steps 5–24). Finally, to generate digoxigenin-labeled RNA
probes, anti-sense and sense DNA templates are transcribed
in vitro from the linearized template with RNA polymerases and
digoxigenin-labeled nucleotides and then purified from the other
components of the polymerase reaction (steps 25–33). Potential
overnight stopping points are after step 4 or step 24.
For our NPY example, the anti-sense probe was generated by
linearizing the cDNA plasmid with EcoRI and transcribing with
T3 polymerase, and the sense probe was generated by linearizing
with NotI and transcribing with T7 polymerase (Fig. 2b).
1. Prepare a restriction digest in 200 ml of volume, containing
160 ml of water, 10 mg of cDNA, and 20 ml of 10× buffer
(provided with enzyme). Vortex for 1 s.
2. Add 50 units of the restriction enzyme (five units of enzyme/mg
of DNA). Do not vortex after the enzymes are added.
3. Incubate for 2 h at the appropriate temperature for your
4. Place tubes on wet ice or at 4°C (or for long-term storage
keep them at −20°C).
5. Place the Eppendorf tubes containing the restriction digest
on wet ice.
221In Situ Hybridization Method for Localizing Gene Expression Changes
6. Add 200 ml of phenol:chloroform:isoamyl alcohol (25:24:1)
7. Vortex for 5 s.
8. Centrifuge at 21,000 × g for 5 min at 4°C. After centrifuga-
tion, the DNA remains in the upper aqueous phase, whereas
most proteins localize in the interface and organic phase.
9. Label a new set of Eppendorf tubes.
10. With a micropipette, collect around 150 ml from the clear
upper phase without disturbing the interface. This fraction
contains the DNA templates. The next step is to precipitate
and concentrate the DNA templates from this fraction.
11. Add 15 ml of 3 M sodium acetate, pH 7.0, at 4°C to the
150 ml of supernatant containing the DNA templates.
12. Add 330 ml of 100% ethanol stored at −20°C. It is important
that the temperature of the ethanol be −20°C when added to
13. Vortex for 5 s.
14. Place the samples at −80°C for 30 min or at −20°C for
15. Centrifuge the samples at 21,000 × g for 20 min at +4°C.
16. The DNA templates will precipitate in a very clear, almost
invisible, white pellet. Remove most of the supernatant (leave
around 10–20 ml).
17. Add 500 ml of 70% ethanol stored at −20°C (do not vortex or
agitate the tubes). The goal is to wash the DNA pellet but not
to resuspend it.
18. Immediately centrifuge the samples, before the pellet resus-
pends, at 21,000 × g for 5 min at 4°C.
19. Remove almost all of the ethanol with a pipette and wait for
2–5 min until the ethanol evaporates. The white DNA pellet
might be difficult to observe.
20. Resuspend the pellet with TE buffer to achieve a final con-
centration of 1 mg of DNA/ml, about 8 ml (see Note 12).
21. Incubate for 1–2 min at room temperature to resuspend the
22. Measure the DNA concentration with a spectrophotometer
and adjust the concentration to 1 mg/ml (optional).
23. For quality control, run 1 mg samples of DNA on a 1% aga-
rose gel containing 0.25 mg/ml of ethidium bromide (see
Note 13). On a piece of parafilm, place a drop of 1 mg of
DNA (1 ml of the purified DNA templates). Add 8 ml of water
and 1 ml of 10× BlueJuice Buffer. Run uncut plasmid (cDNA),
anti-sense template (cDNA + Enzyme A), sense template