In situ Hybridization


Bake all the items mentioned below at 180 deg for over night.

  1. Glass cylinders –Different sizes – 1000ml – 2, 500ml, 100ml, 50ml
  2. Glass bottles (remove the orange cap and the plastic ring and soak it in the 0.1N NaOH)
  3. Spatulas – different sizes
  4. Stir bars-very small sizes for xylenes. Different sizes.
  5. Razer blades.

Keep all the glass items in the Big Oven and turned to Heat to 180 deg.

NaOH washing for plastic Tubwares.

Make 0.1M NaOH solution

NaOH—-4Grams/Liter. Soak all the Plastic Containers in NaOH water for overnight, cover it. Next day wash well with 17.5 megaohm water.

DEPC Treated water.0.8ml of DEPC/Liter

(to make RNAse and DNAse free water)

Make 6 Flasks of 2500ml of water each. DEPC treatment should be done in the Hood.

Add 2.0 ml of DEPC to 2500ml of 17.5 megaohms water. Cover it with parafilm and shake well until DEPC dissolves in water. Then cover with Aluminium foil loosely and shake to wet the rim of the flask and leave it in the hood for Overnight. Next day Autoclave for 30 minutes at 121 degree celsius.

Insitu pre- Hybridization

6X Salts

3.6 mL — 5M NaCl (DEPC treated)

0.6 mL — Tris 1M pH 6.5 (use DEPC water but do not mix DEPC in Tris Buffer)

3.0 mL — 0.2M NaPO4 pH 6.8(DEPC treated)

0.6mL — 0.5M EDTA (DEPC treated)

2.2 mL — DEPC water


10 mL —- Total

Pre – Hybridization Mix for 100 slides ( without formamide)

6X salts —–                           3333.00 uL

Dextran Sulfate(50%)——    4000.00 uL

50X Denhards —————-     400.00 uL

t-RNA (100mg/mL)———— 200.00 uL

DEPC water —————–       567.00 uL

8500.00 uL

Mix everything in the Nutator for 1-2 hrs. Then aliquate into 500 ul into an orange cap screw tubes and stored in -70 0 C freezer. Cut the tip of 1000ul because the pre- Hyb is viscous


Aliquate 1ml of fomamide in 1ml orange screw cap tube and store in –70 .

Final Hybridization Mix.

Mix equal quantity of pre-hybridization and formamide for hybridization. This step you can do at the time of experiment.

50 % Dextran Sulfate ( 40ul–final Volume)

Add 20 gram of Dextran Sulfate (on Sridevi’s cabinet) to 10ml of dd water in a 50 ml conical tube. Add Dextran sulfate little by little because it will make clumps. Put the tube in the rocker/shaker. Add water below 37ml. Leave it in the rocker at least one day or two days. Then make up the volume to 40 ml, cover with tin foil and store it 4 degree celsius.

tRNA (100mg/mL)

Add 250ul of dd water to 25 mg of tRNA powder. (Usually we order 25mg in a vial)

Boeringer Block –10X

Dissolve 25 gms Block powder in 250 mls of1M Tris + 1.5M Nacl2 pH 7.5.

Heat in microwave to 60 deg C. stir until it dissolves. Then aliquate 45 mls in 50 ml conical tubes and freeze in –80 freezer. It will come for 5 experiments.

—— —————

Day-1 Preparations


  1. Turn on 80 deg. heat block
  2. Turn on 55 deg oven for Hybridization

Solutions preparation.


  1. 50mM Tris pH7.5 and 5mM EDTA ( for Proteinase K )

4.5 ml EDTA (0.5M)

22.5 ml. Tris pH 7.5 or pH 8.0 (1M)

423.0 ml ddH2O

Remove 1 ml of this solution to a clean eppendorf tube to make up the proteinase K   solution later. Warm the rest of the solution to 37 deg C in water bath in advance. Tightly seal to prevent contamination.

Proteinase K (This step should do before you incubate slides for proteinase k digestion).

Invitrogen proteinase k- cat # 25530-015.

Take 3 ug = 0.0030 gms and dissolve in 1 ml of Tris/EDTA solution. From that take 45ul into 450 ml of Tris/EDTA buffer.

  1. Paraformaldehyde   solution. (4% in 1X PBS)

350 ml ddH2O

40 ml 10X PBS

heat liquid to approx. 60 deg. C in microwave.

add 16 grams of paraformaldehyde. mix on stir plate in hood

add one chip of NaOH to bring pH up to approx. 11.

let it dissolve and then bring back to room temp. (Can use ice if in a hurry)

Adjust pH with 60 ul of H2SO4 to close to pH 7.0. Check this with pH paper.

  1. 0.2% Glycine in 1X PBS

50 ml of 10X PBS

450 ml ddH2O

Dissolve 1 gram of glycine in 500 ml 1X PBS solution

  1. 1X PBS

50 ml of 10X PBS

  • ddH2O
  1. 0.2x SSC ( 2000mL)

Add 20ml of 20X SSC to 2000mL of DEPC water and place it in the 55 degree celsius oven overnight for tomorrow washes.

  1. Xylenes in 2 glass dishes with micro spin bars. Keep it in the hood.
  2. Ethanol series.
ETOH (95%) except for the first two which are 100% absolute ddH2O NaCl (5M)   time in solution
500   100 % ETOH 1 min.
500   100 % ETOH 1 min.
500   95 % ETOH 30 sec
446 39 14.7 85 % ETOH, plus 0.85% NaCl 30 sec
367 118 14.7 70 % ETOH, plus 0.85% NaCl 30 sec
263 223 14.7 50 % ETOH, plus 0.85% NaCl 30 sec
158 327 14.7 30 % ETOH, plus 0.85% NaCl 30 sec
485 14.7 0.85% NaCl 2 min.
      ddH2O 5 min.
      1XPBS(50ml PBS+450ml of water) 2 min.



  1. Mark slides with date and type of probe going tobe used. Put slides in metal rack (30 slides holders). Incubate in 100% xylenes for 10 min.
  2. Again in 100% xylenes for 10 minutes.
  3. Then follow the Ethanol series.
Series time in solution
100 % ETOH 1 min.
100 % ETOH 1 min.
95 % ETOH 30 sec
85 % ETOH, plus 0.85% NaCl 30 sec
70 % ETOH, plus 0.85% NaCl 30 sec
50 % ETOH, plus 0.85% NaCl 30 sec
30 % ETOH, plus 0.85% NaCl 30 sec
0.85% NaCl 2 min.
ddH2O 5 min.
1XPBS(50ml PBS+450ml of water) 2 min.
  1. Proteinase K Digestion .

Incubate for 30 minutes at 37 deg. C with occasional agitation.

Note: depending on the strength of the individual batch of Proteinase K you may have to titrate this to make sure you got it correct. If the tissue looks really chewed up you used too much for too long, if there is a very weak or no signalat the end of the in situ, one possibility is that you didn’t digest long enough. Unfortunately there are other places where things can go wrong to lead to the no signal result.

Stop proteinase K reaction with a 2 min. wash in 0.2% glycine solution in 1X PBS solution.

2min in 1X PBS – 2 washes

Incubate 10 minutes in 4% paraformaldehyde/1xPBS solution in hood.

5 min. in 1X PBS – 2 washes.

  1. Ethanol series.

Start at 30 % ETOH, plus 0.85% NaCl up to 100% ETOH. 30 sec to 1 min each step. After final 100% ETOH wash (use fresh 100% ETOH for this final wash step) then remove rack and dry a bit on paper towels. remove excess ETOH by tapping bottom of metal rack well against paper towels.

  1. Drying

Incubate for 30 min. in a vacuum drying apparatus. We use a vacuum oven with no heat on. Get the slides totally dry by pulling a vacuum.


Pre- Hybridization preparation.

Thaw prehybridization mix and formamide (aliquate already in orange cap tube) from the –70 freezer. For 30 slides, I usually grab 6 tubes (500ul each) prehybridization mix and 4 (1ml each) formamide tubes.

Mix equal quantity of pre hyb and Formamide and leave on the nutator for 30 minutes.

Probe preparation.

Probe ingredients 1X(1 pair) 5x(enough for 4 pairs )
Dig labelled RNA 1ul 5ul
DEPC water 14ul 70ul
Formamide 15ul 75ul
Total 30ul  


Mix DEPC water, formamide and probe. Denature at 80 degrees C for 2 minutes. Quick cool in ice water bath. Spin down and keep on ice. Amount of probe indicated is approximate, it is good to try 1 microliter per slide pair to start if you had a desent sized band of RNA after the transcription.

Then for each probe pair mix 170 ul of hyb mix for each 30 ul of probe/formamide mix. Mix well by inversion. This stuff is viscous and must be mixed well before applying to the slide pairs. try to invert in for several minutes to mix without adding bubbles to the solution. Use pipet tip to mix well

Hybridization mix One slide pair
Pre Hyb/Formamide mix 170 ul  
Probe 30 ul  
Total 200 ul  

Take a pair of slides. Add 100- 130 microliters Hybridization mixture to the top of each slide (200 – 260ul microliters per pair). Spread out across the dried tissue sections with the side of a clean micropipette tip. Use surface tension to avoid disturbing the tissue sections. Once all the tissue sections are wet. Hold the two slides upright so that the solution starts to fall to the bottom edge of the slide. Hold the two bottom edges of the slides touching each other at the raised white triangles (on the “probe-on-probe plus” slides). do this while resting the bottom of the slides on top of a piece of parafilm to prevent the probe from running off the slides. Slowly move the slides together creating a sandwich and allowing the probe to squeeze up between the slides, covering the tissue sectios without generating too many air bubbles. This takes a bit of practice. There is no need to seal the edges of the slide sandwich with rubber cement. Moist environment of hyb. Box will keep slides wet.

Change gloves between probes.

Place slide pairs on homemade plastic pipet rack inside tupperware box.

Seal tupperware with parafilm to make sure it stays moist.

Hybridize for Overnight to 36 hrs at 56 deg C. Use the most constant temp incubator you can find.

Note : Don’t forget the make 0.2X SSC ( about 2000ul) and keep it in 55 degree C. Keep 2 Glass staining dishes and slide metal rack at 55 deg C too.


Day 2- Washing steps

Washing with 0.2X SSC.

Separate slides by dipping in o.2X SSC. Change SSC between probes. Put slides on metal rack and keep it in 0.2X SSC at 55 deg C in an oven with rocking platform.

In Situ Step by Step- Day 2



0.2X SSC 60 min at 55 degree C in an oven with agitation Prepare for 2000 ml

20 ml of 20x SSC

1980 ml of DEPC water

0.2X SSC 60 min at 55 degree C in an oven with agitaion  
Boeringer Block 45 min with agitation at RT 45 ml of 10x stock( grab from –80 and thaw)

400 ml of DEPC water

TBNT 45 min with agitation at   RT

Prepare for 2500ml

25 gms of BSA

250 ml Tris/Nacl stock pH7.5

750 ml of 10% Triton X

2150 ml of DEPC water

1:1250 (antibody:TBNT )


Place slide in slide mailers five per mailer. Fill the mailer with 12ml of ab:TBNT soln. Prepare for 2 batches(38ml in each) Total about 76ml.

38 ml of TBNT

31 ul of Dig oxigenin

TBNT 20 minutes with agitation  
TBNT 20 minutes with agitation  
TBNT 20 minutes with agitation  
TBNT 20 minutes with agitation  
100mM Tris pH 9.5;

150mM Nacl

50mM Mgcl2

15 Minutes with agitation

Prepare for 1500 ml

150 ml of 1M Tris/1.5Mnacl,

150 ml of 0.5M of Mgcl2(open in hood)

1200 ml of DEPC water

100mM Tris pH 9.5;

150mM Nacl

50mM Mgcl2

15 Minutes with agitation  
100mM Tris pH 9.5;

150mM Nacl

50mM Mgcl2

15 Minutes with agitation Make sure all the detergent has washed off.
Western blue Overnight in dark. Place slides in clean plastic slide mailers and fill these with Western blue from promega. Need about 12mls for each mailer.
Check under scope for development on next day morning.


Insitu – Final step

Stoping western blue reaction with TE (10mM Tris pH 7.5 &1mM EDTA pH8.0)

Make 500ml of TE for first wash.

1M Tris pH7.5    ———-              5 ml

0.5M EDTA pH8.0 ——-             1 ml

deionised water ———- –          494 ml


Total                                                500 ml


Washing 1. wash with TE for 10 minutes

Washing 2. wash with deionised water for 5 minutes without agitation.

Mounting. Drain the excesss water on the paper towel. Then ,add 4 drops of Aqua poly mount on the slides and cover with coverslip.






STOCK NAME COMPONENT M.W. GRAMS Preparation and sterilization
20 X SSLC -1L 3 M Nacl 58.44 175.3 gms DEPC treatment and   autoclave
pH 7.0 300 mM Na Citrate 294.1 88.2 grm  
10X PBS – 1L 1.3 M Nacl 58.44 75.97 gms DEPC treatment and   autoclave
pH 7.0 70 mM Na2HPo4 141.96 9.93 gms  
  30 mM NaH2Po4 137.99 4.13 gms  
0.5M EDTA – 1L 0.5 M EDTA 372.24 186.1gms use NaoH to dissolve EDTA (about 18 gms)
p.H 8.0       DEPC treatment and   autoclave
0.5M Mgcl2 – 1L 0.5 M Mg cl2 203.3 101.65gm DEPC treatment and   autoclave
5M Nacl -1L 5 M Nacl 58.44 292.2 gm DEPC treatment and   autoclave
1M Tris/1.5M Nacl      
pH 7.5 1M Tris HCl 157.6 157.60gms initial pH reading was 4.15
1L 1.5 M Nacl 58.44 87.66gms use NaoH pellet about 100 to bring pH 7.5
        autoclave . No DEPC treatment
1M Tris/1.5M Nacl        
pH9.5 1M Tris free base 121.14 121.44 gms Initial pH readings was 10.34
1L 1.5M Nacl 58.44 87.66 gms Use HCL to bring down pH 9.5
        autoclave. No DEPC treatment
Denhards 50X PVP   0.5 g  
  Ficon400   0.5 g  
  BSA (Fraction V)   0.5 g bring up to 50 ml with DEPC water

·       DEPC Treatment:

Use 0.8 ml of DEPC to each 1 litre of solution and shake well with parafilm in the chemical hood
Then covered with Aluminium foil loosely for overnight. Make sure the rims are wet with the solution.


Chemicals for in situ hybridization

Name Vendor Cat. No. Price($)
ApaL I BioLabs R0507S 58
Nco I Promega R6513 81
4-CORE® Buffer Pack (Buffers A, B, C, D) Promega R9921 10
Riboprobe® System – SP6 Promega P1420 133
Riboprobe® System – T7 Promega P1440 133
RNase free DNase Promega M6101 45
DIG RNA Labeling Mix Roche 11277073910 146
Anti-Digoxigenin-AP Roche 11093274910 200.00
Blocking Reagent Roche 11096176001 114.00
Ficon 400 Sigma-Aldrich F2637-5G 29.60
formamide Sigma-Aldrich F9037-100ML 44.70
PVP Sigma-Aldrich 234257-5G 49.20
PVP EMD 529504-100GM  
BSA, Fraction V EMD 2930 177.00
DEPC EMD 3660 154.00 × 2
Xylenes Fisher X3P-1GAL 116.46
Western Blue Fisher PR-S3841 48.46 × 2
Dextran Sulfate Fisher BP1585-100 246.86
Glycine Sigma G7126-100g 20.30
Proteinase K Invitrogen 25530-015 96.00
tRNA Invitrogen 15401-029 127.00
paraformaldehyde Ted Pella, Inc 18501 22.90
100% ethanol pharmci-aaper 111ACS200  
Aqua poly/Mount Polysciences, Inc. 18606-20 60.00
Triton X-100 Fisher BP151500 46.12
0.375% Elmer’s Carpenter’s Wood Glue      

Facilities for in situ hybridization

Name Vendor Cat. No. Price($)
Probe Microscope Slides Fisher 22-230-900 80.98
Oven (for hybridization) Precision, Thelco   About 1500
Oven (for paraffin) Curtin Matheson Scientific, Inc.    
Microwave oven BioWavePro, PELCO    
Microscope Nikon ECLIPSE E600    
Tissue Imbedding Capsules Fisher 15-182-218  
Paraffin SPI 2846A-AB  
Plastic frames      
Megohms-CM 75 II MyronL Company B2-VW-E3452 220.28
Rotator Barnstead Lab-Line   About 800
Stir plate Nuova ll Thermolyne    
Coverslips Fisher 12-543B 33.87
Coverslips Fisher 12-543D 33.67
Coverslips BioExpress 2935-246-G  
Coverslips VWR 48382-139 139.89
RNase free tips      
RNase free 1.5ml tubes      
Clat Adams Nutator VWR 15172-203 633.89
Glass cylinders – 1000ml VWR 89000-276 72.24 × 4
Glass cylinders –500ml VWR 89000-274 66.45 × 4
Very small size stir bar VWR 58948-411 6.92 × 2
Small size stir bar VWR 58948-138 8.69 × 10
Medium size stir bar VWR 58948-150 10.80 × 10
Large size stir bar VWR 58948-171 13.02 × 10
Very large size stir bar VWR 58948-309 13.37 × 5
Razer blades VWR 55411-055 27.59
pH indicator strips VWR 60772-001 33.69
Glass flasks – 3000ml OnlineScienceMall FLA206 42.95 × 6
Glass dishes Ted Pella, Inc 21054 69.90× 7
Metal rack Ted Pella, Inc 21072 48.90× 2
Slide mailer VWR 82003-430 14.81
Plastic chamber Walmart   × 2
Plastic box Walmart   × 30
Big plastic containing holding 20L Walmart   × 1