In situ hybridization to mRNA using DIG labelled riboprobes (for
cryosections), with optional tyramide amplification
Mikael Niku 3.4.2006, version 1.5
Dept. Basic Veterinary Medicine, University of Helsinki, Finland
The protocol is optimized for bovine tissues . The procedure
takes 3 days.
solutions clean. This is the most sensitive step in the whole
procedure!
We use standard DIG-labelled riboprobes, prepared with in
vitro transcription and purified by LiCl-ethanol precipitation
or Sephadex columns.
Dilute the probe in the hybridization buffer. Determine
optimal concentration empirically. We typically store a
standard 20 l transcription reaction in 100 l 50%
formamide and dilute this 1/100.
The homemade tyramide conjugates are prepared according
to Hopman et al (1998) J Histochem Cytochem 46: 771-777.
We recommend Superfrost Plus slides to keep the tissues
firmly attached.
We currently use Shandon Coverplates for posthybridization detection steps. They make life a lot easier, but
identical results can be obtained without them, by using a
PAP pen and a humid chamber. In this case, be very careful
to perform all humid chamber incubations on an exactly
level surface, in order to avoid drying sections.
Recipes for solutions are included in the end of the text. We
don’t use DEPC, but select high quality reagents and use
fresh milliQ water.
1. Preparing the sections for ISH
Cut fresh 10 m cryosections from tissue samples stored in
-80C (preferably less than 6 months old), on Superfrost
Plus slied.
Heat the slides on a 60C block for 1 min. Air dry at room
temperature for 1 hour.
Fix the sections in ice-cold 4% paraformaldehyde (PFA) in
PBS buffer, pH 9.5, for 1 hour (alkaline fixation will
enhance the detection).
Wash (PBS, 5 min).
Incubate in 0.2 M HCl for 6 min.
Wash.
Denature the probe: 85C, 5 min.
Apply 100 l / slide and cover with a coverslip (turn the top
side during drying against the sample). Pipet a continuous
strand near one long edge of the slide, then carefully lower
the coverslip on the sample, avoiding any bubbles. If you get
bubbles anyway, try to remove them by carefully moving the
coverslip around. Do not press!
Pack slides into hybridization containers (small slide
boxes with tight lids). We recommend sterilizing the boxes
(e.g. with flame). Include a piece of tissue paper etc.
dampened with 4 x SSPE, 50% formamide. Close the
containers and use plastic adhesive tape to ensure they are
airtight.
Incubate slides overnight at optimum hybridization
temperature (typically 50-55C).
3. Washes and antibody
Transfer the slides from the containers into 4 x SSPE in
racks. Incubate 10-15 min. The coverslips should drop; if
they don’t, gently help with forceps. Check that all coverslips
come off!
Wash:

4 x SSPE / 50 % formamide 10 min at hybridization
temperature

Quick rinse in 4 x SSPE

1.5 x SSPE 10 min at hybridization temperature
Incubate in 0.5 – 2 g/ml proteinase K in 20 mM TrisHCl pH 7.4, 2 mM CaCl2, for 30 min at room temperature.
Transfer slides into MA buffer. Pack into coverplates.
Wash.
Block: apply 250 l 2 % blocking solution per slide. Incubate
30 min (coverplate racks with lids on).
Optionally, postfix in 4% PFA for 5 min. You might need
this for high hybridization temperatures; otherwise, it can
be better to skip this.
For tyramide amplification only:
Dehydrate: transfer the slides gradually to 100%
ethanol (30% ethanol:PBS, 50% ethanol:PBS, 75%
ethanol: H2O, 85% ethanol, 95% ethanol, 100%
ethanol; quick rinse in each except 5 min in 75%).
Let dry on blotting paper (about 30 min).
Rinse coverslips in 100% ethanol and let dry on
clean tissue paper 30 min.
2. Hybridization
Note that the hybridization solution contains formamide, so
work in the hood. Also take care to keep the slides and

Incubate in anti-DIG-POD conjugate, diluted
1:100 – 1:300 in 2% blocking solution (determine
optimal concentration empirically), 1 h at room
temperature, with Coverplate rack lids on.

Wash in MAT buffer (MAB + 0.1% Tween-20).

Wash in PBT buffer (PBS + 0.1% Tween-20).

Incubate in DIG-labelled tyramide (from a
commercial kit, according to manufacturer’s
instructions, or homemade tyramide in PBS + 0,1 M
imidatsoli + 0,001 % fresh H2O2, pH 7.6, for 10
min.
1

Wash in PBT.

Wash in MAT.
Apply anti-DIG-AP: dilute 1:1000 in 2 % blocking
solution. Apply 100 l / lasi. Incubate 2 hours at room
temperature, with lids on.
Wash twice with MAT buffer.
4. Color reaction
Wash: basic detection buffer 5 min.
Dilute NBT/BCIP chromogen, 1:50 in basic detection buffer. (NBT/BCIP is best stored at -20C, but tends to precipitate. Thaw in warm water bath, mix with a pipette until resolubilized).
Apply 250 l / slide. Incubate o/n at room temperature (in
some cases, overnight at +4C or 2 x overnight at room temperature might be better).
Wash: basic detection buffer 5 min.
5. Counterstaining and embedding
Transfer slides from coverplates into water in racks. Counterstain in hematoxylin, etc.:

Mayer’s hematoxylin 30 s.

Warm running water 5 min.
Embed with Faramount, let dry, and enjoy!
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Reagents
PBS buffer (phosphate buffered saline)
10 x PBS (1 l / 4 l):
80 g / 320 g
2g/8g
14.4 / 57.6 g
2g/8g
NaCl
KCl
Na2HPO4
KH2PO4
Adjust pH with NaOH -> 7.4 (originally about 6.7).
Autoclave. Use as 1x dilution. Do not use unsterilized diluted buffer for ISH for longer than a few days.
Consumption in ISH with coverplates:

PBS: n. 500 ml / 20 slides

PBT: n. 100 ml / 20 slides
SSC buffer (saline sodium citrate) pH 6
20 x SSC pH 6 (1 l):
175.3 g NaCl (= 3 M)
88.2 g Na3sitraatti2H2O (=0.3M)
Adjust pH  6.0 with concentrated HCl. Autoclave.
Use in microwave treatment as 2x dilution.
Consumption in ISH: 500 ml / 20-40 lasia.
Tris-HCl buffer
1 M Tris-HCl (1 l / 4 l):
121.14 g / 242.28 g Tris
Adjust pH with conc. HCl (4 l pH 7.4: about 140 ml; pH 9.5:
a lot less). Settles slowly – let stay overnight, check again
and readjust. Autoclave.
1,4 ml
1,4 ml
50 x Denhardts
H2O
Heat 80C/10 min, cool on ice, store at +4C. Should be OK
for several weeks.
Consumption in ISH: 100 l / slide.
SSPE buffer
20 x SSPE pH 7,4 (1 l / 4 l):
175.3 g / 701,2 g NaCl
27.6 g / 110,4 g NaH2PO4H2O
40 ml / 160 ml
0.5 M EDTA
Adjust pH with 10 M NaOH:lla. Autoclave.
MA buffer (maleic acid), pH 7.5
10 x MAB buffer (1 l / 4 l):
116.1 g / 464,4 g maleic acid
87.66 / 350.64 g NaCl
Adjust pH with NaOH (4 l: n. 310 g + 40 ml 10M NaOH
solution) – maleic acid solubilizes at about pH 6.7 (RT). pH
changes slowly first; after solubilization, easily jumps.
Use at 1 x dilution.
Consumption in ISH (coverplates): 200 ml / 20 slides +
MAT 2.5 ml / slide.
NaCl
5 M NaCl (1 l):
292.2 g NaCl
Autoclave.
EDTA
Blocking solution
0.5 M EDTA (1 l):
10% blocking solution (100 ml):
186.1 g EDTA (Na2, 2H2O)
10 g
Roche Blocking Reagent
100 ml 1 x MA buffer
Adjust pH with 10 M NaOH  8 – doesn’t solubilize below
this. Autoclave.
PFA
4 % PFA/PBS (ready-to-use):
Solubilize 4 % paraformaldehyde (w / w) in PBS by heating
in 60C water bath for 1-2 h with occassional swirling.
Freeze, do not refreeze.
Consumption in ISH:ssa: 500 l / slide (in coverplates).
Hybridization buffer
1 x hybridization buffer (14 ml):
7 ml
2.8 ml
1,4 ml
deionized formamide (store at +4C)
20 x SSPE
10 mg/ml ssDNA
Solubilize by heating at 60-65C water bath. Mix often by
vigorous agitation. Will solubilize in about 15-30 min, will
remain cloudy. Cool, aliquot and freeze. Melt in a warm
water bath (tends to precipitate in the cold). Should be OK
for about two weeks at +4C.
Use as 2% dilution. Consumption in ISH: 350 l / lasi.
Basic detection buffer
Basic detection buffer (100 ml, ready-to-use):
10 ml
2 ml
5 ml
83 ml
1 M Tris-HCl pH 9.5
5 M NaCl
1 M MgCl2
H2O
We prepare this fresh from component solutions, but could
be prepared as a 10X stock, autoclaved, and stored.
Consumption in ISH (with coverplates): 6 ml / slide.
3
MgCl2
1 M MgCl2 (1 l):
Commercial reagents
203.3 g MgCl2  6H2O
Autoclave.
Blocking solution: Roche 1 096 176
Anti-DIG Fab: Roche 1 093 274
NBT/BCIP: Roche 1 681 451
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