Zhu Lab In Situ Hybridization Protocol (paraffin sections)
Hybridization (First day of ISH protocol)
1. Warm-up slides on slide warmer at 58oC for 1 hour.
2. Meanwhile, IN THE HOOD make 4% paraformaldehyde (PFA) in PBS as
follows:
A. Heat 200 mL ddH2O (use “express 1” setting on microwave, 2X)
B. Put on hot plate, add stir bar, and stir to measure temperature (should be
60oC-70oC)
C. Measure 16g paraformaldehyde and put in the ddH20
D. Add 8-10 μL of 10N (10M) NaOH to help dissolve the solid PFA
E. Add 3g of NaCl and 100 mL of 0.4M PB solution
F. Bring volume up to 400 mL with ddH2O and chill on ice
3. Cool slides down to RT, then dewax and dehydrate as follows:
A. Xylene wash for 3 min, 3 times
B. 100% EtOH for 1 min, 3 times
C. 95%, 70%, 50%, 30% EtOH series, 1 min each
D. DEPC-PBS for 1 min
4. Fix slides in fresh 4% PFA-PBS for 30 min at RT
5. DEPC-PBS wash for 5 min, 2 times
6. Digest with proteinase K (2 μg/mL, use 20 μL of a 20 mg/mL stock solution) in
200 mL proteinase K buffer for 10 min at RT.
NOTE: If performing ISH on both paraffin and cryostat sections, use 2 separate
staining tanks, one with 20 μL of proteinase K (paraffin sections) and one with
10 μL of proteinase K (cryostat sections)
7. Fix slides in 4% PFA for 10 min at RT
8. Wash in DEPC-PBS for 5 min with stirring on a stir plate
9. Submerge slides in freshly made acetylation solution (as described below) for 10
min at RT
1. 200 mL DEPC-H2O
2. Take 3 mL out
3. Add 2.4 mL of dissolved triethanolamine and mix thoroughly by
pipetting
4. Add slides
5. Add 520 μL of acetic anhydride and mix by pipetting
10. Wash slides in DEPC-PBS for 5 min, 2 times
11. Meanwhile, make a humidified hybridization chamber (line tin with wet paper
towels, place grid inside)
12. After final PBS wash, aspirate off most but not all of the liquid from around the
sections and dry the bottoms of the slides with Kimwipes
13. Place the slides on the grid in the humidified chamber
14. (Pre-hybridization) Add hybridization solution (1 mL/slide) directly onto the
slides making sure to cover each section with hybridization solution. Place lid on
the tin and incubate in hybridization incubator at 65oC for 1.5 hours (minimum 1
hour up to a maximum of 4 hours)
15. Meanwhile, add moist paper towels to a microscope slide box to make a
humidified chamber.
16. Aspirate off as much liquid as possible from around sections, dry bottom of
slides, and add probe (1:10 dilution of probe in hybridization buffer) to each
section on the slides (use ~ 100 μL/slide). Use a needle to slowly and gently
lower the coverslip onto the slide, being sure to eliminate as many air bubbles as
possible. Place slides into the slide box positioned horizontally so that the slides
lay flat (parallel to benchtop). Completely seal sides of box with tape, then
incubate at 65oC overnight
Washes and antibody staining (Second day of ISH protocol)
1. Pre-warm 5X SSC (50 mL 20X SSC in 150 mL dH2O) and 0.2X SSC (200 mL
dH2O, remove 2 mL, add 2 mL 20X SSC; two separate tanks) to 65oC (or
whatever temperature at which the slides were incubated overnight) in staining
tanks in the hybridization incubator (about 15-20 minutes).
2. Take boxes of slides out from oven and place slides in a slide carrier.
3. Put slides into tank in hybridization incubator containing 5X SSC for 5 min at
65oC
4. Using forceps, remove slides one by one from the slide carrier, agitating the slides
as they are removed to loosen/remove the coverslip. Remove coverslips that fall
into the tank and replace the uncovered slides back in the slide carrier.
5. Put slides into tank with 0.2X SSC in hybridization incubator for 30 min at 65oC,
2 times
6. Put slides into 0.2X SSC at RT for 5 min
7. Transfer slides to buffer B1 for 5 min at RT, with stirring
8. Aspirate off as much liquid as possible from slides, wipe bottom of slides with
Kimwipe, then place on grate inside humidified staining chamber (foil-coated
chamber)
9. Place 1 mL of buffer B2 (100 μL normal goat serum, NGS, per 10 mL Buffer B1;
make enough for 2 mL per slide) on slides and incubate in humidified staining
chamber for one to several hours at RT
10. Aspirate off liquid from around sections and dry back of slides with Kimwipes.
Place slides back in humidified staining chamber and add 1 mL of a 1:4000
dilution of anti-digoxygenin antibody in Buffer B2 to each slide (2.5 μL antibody
in 10 mL Buffer B2). Incubate at 4oC overnight
Detection (Third day of ISH protocol)
1. Wash slides in buffer B1 at RT for 10 min with stirring, 3 times. To reduce
background staining, more washes may be performed.
2. Make 250 mL of fresh buffer B3 (200 mL for equilibration of slides, 50 mL for
making buffer B4) and equilibrate slides in buffer B3 for 5 min

25 mL 1M Tris, pH 9.5

5 mL 5M NaCl

12.5 mL 1M MgCl2

dH2O to 250 mL
3. After equilibration, dry the backs of the slides and partially dry the sections on the
front of the slides, making sure not to dry out the sections
4. Prepare ≥ 500 μL buffer B4 per slide (as described below), then overlay slides
with ≥ 500 μL buffer B4 in the slide staining rack.

For every 1 mL of Buffer B3, add 4.5 μL NBT, 3.5 μL BCIP, and
10 μL levamisole (400mM stock)
5. Incubate at RT from at least 2 hours up to 48+ hours, depending on probe and
type of section (paraffin sections require longer incubation times )
6. Stop reactions by washing slides in TE, pH 8.0 at RT for 5-10 min, repeating with
fresh TE if needed
7. Meanwhile, put DakoMount in 50oC water to liquefy.
8. Aspirate off most of the liquid from the slides, then completely dry the slides on
the slide warmer set at 37oC for several minutes
9. Add one drop of DakoMount to each section on the slide, then add a coverslip,
being very careful not to introduce air bubbles
10. Allow slides to sit at RT for at least 20 minutes to allow mounting medium to
harden
11. Once slides have dried/mounting media has hardened, check slides under
microscope to verify that no air bubbles have been introduced over the sections.
12. If air bubbles are present, heat up TE buffer (pH 8.0) to 50-60oC. Place slides to
be remounted in slide holder and place in TE for several minutes. Remove slides
as soon as the coverslips have fallen off.
13. If the coverslips have not fallen off after several minutes, remove slide holder
from TE and individually dip slides in the tank, agitating the slides gently to
facilitate the removal of the coverslip. DO NOT REMOVE COVERSLIPS BY
HAND AS THIS MAY SCRATCH/DAMAGE THE SECTIONS!!!
14. Dry backs of slides and place on slide warmer set at 37oC for several minutes.
Re-mount coverslips (Steps #9-11, above).
Zhu Lab In Situ Hybridization Protocol (frozen sections)
Hybridization (First day of ISH protocol)
1. Transfer slides from -80oC to RT for 5 min, then place on slide warmer set at
50oC for 30 minutes.
2. Meanwhile, IN THE HOOD make 4% paraformaldehyde (PFA) in PBS (as
described above for paraffin sections)
3. Fix slides in 4% PFA-PBS for 10 minutes.
4. Wash slides with DEPC-PBS for 5 min, twice
5. Digest tissue in proteinase K (1 μg/mL) for 15 minutes (10 μL of 20 mg/mL stock
proteinase K in 200 mL PK Buffer)
NOTE: THIS STEP MAY BE OMITTED (prevents disruption of tumor
mass within the normal tissue)
6. Fix slides again in 4% PFA for 15 minutes.
7. Wash twice with DEPC-PBS, 5 min each time and then wash once with DEPCddH2O alone for 5 min.
8. Submerge slides in freshly made acetylation solution (as described above for
paraffin sections) for 10 min
9. Wash twice in DEPC-PBS, 5 min per wash
10. (Pre-hybridization) Drain off PBS and add hybridization solution (1 mL/slide).
Cover with coverslip and incubate at 65oC for 1.5 hours
11. Take off the coverslip and add probe (~ 100 μL/slide). Cover with coverslip and
incubate at 65oC overnight
Proceed with second and third days of ISH protocol as outlined for paraffin sections
A Note About Reagents and Glassware
The following solutions can be made in untreated glass bottles/flasks. Add 0.05% DEPC
overnight and then autoclave:
EDTA, NaCl, MgCl2, NaHCO2/Na2CO3, NaOAc, 20X SSC, PBS, PB
The following solutions cannot be autoclaved. Make these with DEPC-H2O in sterile
bottles/flasks/glassware:
Tris, SDS, and any buffers/buffer solutions containing Tween, Triton, or CHAPS
The following solutions cannot be autoclaved. Make in sterile 50 mL tubes using only
reagents that have been treated with DEPC or solutions made with DEPC-H2O:
Hybridization buffer, Denhardt’s Reagent
The following solutions do not need to be RNAse-free:
NBT, BCIP, levamisole
Solutions for ISH
0.4M Phosphate Buffer (PB)
165.3g Na2HPO4∙7H2O
25.6g NaH2PO4∙H2O
DEPC-H2O to 2L
87.6g Na2HPO4 (anhydrous)
22.3g NaH2PO4 (anhydrous)
DEPC-H2O to 2L
43.8g Na2HPO4 (anhydrous)
11.15g NaH2PO4 (anhydrous)
DEPC-H2O to 1L
4% Paraformaldehyde in 0.1M PB
Heat 200 mL DEPC-H2O in microwave until hot (NOT boiling), ~ 60-70oC
Add 16g paraformaldehyde and 8-10 drops of 10N NaOH
Stir until dissolved (can take 10-30 minutes), adding more NaOH if needed
Add 100 mL of 0.4M PB solution
Check pH with paper, adding drops of HCl to bring solution pH to 7.0-7.5
(For tissue preparation step only) add 3g NaCl and let dissolve
Bring volume up to 400 mL with DEPC-H2O
0.2 μm filter solution and chill on ice before using
PBS (0.1M PB, 0.15M NaCl) (make at least 2-2L batches)
For 2L:
500 mL 0.4M PB
17.5g NaCl
DEPC-H2O to 2L
Proteinase K Buffer (1 μg/mL proteinase K, 5mM EDTA, 50 mM Tris pH 7.5)
For 400 mL:
5 mL 0.5M EDTA, pH 8
20 mL 1M Tris, pH 7.5
40 μL of 10mg/mL proteinase K
DEPC-H2O to 400 mL
For 300 mL:
3 mL 0.5M EDTA, pH 8
15 mL 1M Tris, pH 7.5
30 μL of 10mg/mL proteinase K
DEPC-H2O to 300 mL
30% Sucrose in 0.1M PB
For 500 mL:
125 mL 0.4M PB
150g sucrose
DEPC-H2O to 500 mL
100X Denhardts Reagent
For 500 mL:
10g Ficoll (Type 400)
10g polyvinylpyrrolidone
10g Bovine Serum Albumin (fraction V)
DEPC-H2O to 500 mL
Filter and store at -20oC
20X SSC
For 1L:
175.3g NaCl
88.2g Na Citrate
dissolve in 800 mL dH2O
bring pH to 7.0
bring up to 1L with dH2O, add 500 µL DEPC, then autoclave
OR
Add SSC powder to sterile bottle and add 1L DEPC-H2O
Hybridization Solution (50% formamide, 5X SSC, 5X Denhardts, 250 μg/mL yeast
RNA, 500 μg/mL herring sperm DNA)
For 500 mL:
250 mL formamide
125 mL 20X SSC
25 mL 100X Denhardts Reagent
0.125g Torula yeast RNA
0.25g herring sperm DNA
store at -20oC
Buffer B1 (0.1M Tris 7.5, 0.15M NaCl)
For 2L:
200 mL 1M Tris, pH 7.5
60 mL 5M NaCl
DEPC-H2O to 2L
Buffer B2 (Buffer B1 with 1% goat or sheep serum)
***Make at least 2 mL per slide for both blocking and antibody incubation steps***
# of slides * 2 * 1 mL = volume of Buffer B1
# of slides * 2 * 10 μL = volume of normal goat serum (NGS)
Buffer B3 (0.1M Tris pH 9.5, 0.1M NaCl, 50mM MgCl2)—MAKE FRESH!
For 500 mL:
50 mL 1M Tris, pH 9.5
10 mL 5M NaCl
25 mL 1M MgCl2
DEPC-H2O to 500 mL
Buffer B4 (Buffer B3 with NBT, BCIP, and levamisole)—MAKE FRESH!
***Make at least 500 μL per slide***
Per mL Buffer B3:
4.5 μL NBT
3.5 μL BCIP
10 μL levamisole (400mM stock)
1X TE Buffer, pH 8.0
For 500 mL:
5 mL 1M Tris, pH 8.0
1 mL 0.5M EDTA, pH 8.0
DEPC-H2O to 500 mL
Other Solutions (DEPC/autoclaved or made w/ DEPC-H2O)
0.5M EDTA, 1M Tris (pH 7.5), 1M Tris (pH 9.5), 5M NaCl, 1M MgCl2