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Gaido Group Protocols

Part 2 of 3 (previous | next)

GUDMAP – SOP “In Situ-Hybridization”
(CIIT at the Hamner)

Last update: March 19, 2007
For questions and comments, please contact:
gaido@thehamner.org     Kevin Gaido
Updated By:  Janan Hensley

Preparation for the ISH run

Waterbaths, incubators, & heatblocks must be turned on to allow time to warm to desired temperature. Remove solutions from 4ºC storage (Formamide, Peroxide, 1X MWB) and equilibrate to RT before using.

DAY 1 continued: Daily solutions

ISH daily solutions
Total volume
  To be heated - 65ºC for 30 minutes before filter and degas steps
1.  Make sure waterbath is at 65ºC.
To be filtered – use whatman paper
2.  Dry slides for 5 minutes at 37ºC.
To be degassed
  To be added last

FIRST DAY SOLUTIONS

Solution Amount/units

Reagent

  catalog # NOTES
1X PBS 150 mL 1X PBS DEPC treated      
750 µL

10% Tween

   
150 mL Total volume    

 

0.2M HCl 5 mL

2M HCl

 

Fisher: A144-212

 
250 µL

10% Tween

   
44.75 mL Ultra Pure DEPC H2O    
50 mL Total volume    

 

5X SSC

preheat to 65ºC

74.5 mL

Ultra Pure DEPC H2O

 

 

 
25 mL

20X SSC

 

Ambion: 9763

500 µL 10% Tween    
100 mL Total volume    

 

0.1X SSC

preheat to 65ºC

99 mL

Ultra Pure DEPC H2O

 

 

 

500 µL

20X SSC

 

Ambion: 9763

500 µL 10% Tween    
100 mL Total volume    

 

Formamide I

preheat to 65ºC

28 mL

Ultra Pure DEPC H2O

 

Roche 11814320001

Bring to RT before using

 

7 mL

20X SSC

 

 

350 µL 10% Tween    
35 mL formamide    
70 mL Total volume    

 

Formamide II

preheat to 65ºC

31.5 mL

Ultra Pure DEPC H2O

 

 

 

 

3.5 mL

20X SSC

 

 

350 µL 10% Tween    
35 mL formamide    
70 mL Total volume    

 

1X NTE

preheat to 65ºC

20 mL

5X NTE

 

 

 

500 µL

10% Tween

 

 

79.5 mL Ultra Pure DEPC H2O    
100 mL Total volume    

 

20mM iodoacetamide (added at end of 1st day)

Degas without IDO, add IDO after degas step

185 mg

iodoacetamide (added last)

 

Sigma I-1149

IDO not stable, add last

Take 50mL 1X NTE from solution made in previous step

50 mL

1X NTE

 

 

- 10% Tween (already in 1X NTE)    
50 mL Total volume    

 

1X TNT

20 mL

10X TNT

 

 

 

 

1 mL

10% Tween

 

 

179 mL Ultra Pure DEPC H2O    
200 mL Total volume    

 

4% sheep serum

(Blocking solution)

filter, degas

add at end of 1st day

     

 

Sheep serum is aliquoted into 5mL aliquots and stored in -20ºC

 

2.4 mL

sheep serum

 

Gibco 16070-096 or Sigma S-2263 (100mL)

6 mL 10X TNT    
300 µL

10% Tween

   
51.6 mL Ultra Pure DEPC H2O    
60 mL Total volume    

 

TNB

preheat to 65ºC

Degas

7 mL 10X TNT  

 

Add blocking reagent before heating so it dissolves into solution

Degas after heating

350 µL

10% Tween

 

 

63 mL Ultra Pure DEPC H2O    
0.35 g

blocking reagent

 

Perkin Elmer

70 mL Total volume    

 

1X Maleate wash buffer

(MWB)

prepare fresh weekly

99.5 mL

1X MWB

 

 

Stored at 4ºC, bring to RT before using.

500 µl

10% Tween

 

 

100 mL

Total volume

   

 

1% BR

preheat to 65ºC

filter, degas

0.5 g

blocking reagent

 

Roche: 11096176001

Stored at -20ºC

Add at end of 1st day

50 mL

1x maleate wash buffer

 

 

50 mL

Total volume

   

 

TMN

94.5 mL

TMN

 

 

 

 

 

filter

500 µl

10% Tween

 

 

prepare levamisole 10mg/mL

5 mL

levamisole 10mg/mL

   
 

100 mL

Total volume

   

 

pre-HYB

15 mL

Formamide

 

 

 

7.5 mL

20X SSC

 

 

 

7.5 mL

Ultra Pure DEPC H2O

   

 

15 µl

tRNA 10mg/ml

 

Ambion # 7119

Stored at -20ºC

15 µl

Heparin 100mg/ml

 

Sigma: H-3149

Stored at -20ºC

30 mL

Total volume      

 

In situ hyb mix

15 mL

In situ hyb mix

 

Ambion B8807G

 

heat at 65ºC (10 minutes)

enough for 12 slides

 

 

 

 

cool to RT

         

add 15µl 100mg/ml DTT

       

Stored at -20 ºC

add 15µl 10mg/ml tRNA

        Stored at -20 ºC

 

Probe

vortex solution, heat at 80ºC for 30 seconds (heat block), then heat at 65ºC for about 10 minutes (waterbath), cool, add to rack

 

300 ng/mL

Probe

 

 

 

quick spin after heating

1 mL

in situ hyb mix

 

 

1 mL

Total volume    

 

Anti-digoxigenin-POD

(1:500)

Add 1mL autoclaved water for a new kit

Add antibody at end of first day

8.5 mL TNB - see note at right

 

Take from TNB

700µl x 12 slides = 8.4mLs TNB (plus 100µl extra) = 8.5mL

Anti-DIG(1:500) = 8.5mL/500 = 17µl

-

10% Tween

  In TNB already

17 µl

anti-digoxigenin-POD

 

Roche: 11207733910

8.517mL

Total volume    

 

0.7% H2O2

Add at last moment

1.16 mL

H2O2

 

Sigma: H-0904

Stored at 4ºC, bring to RT before using.

50 mL

MeOH

 

Fisher: A412-4

 

DAY 2 SOLUTIONS

TSA (tyramide-biotin) (1:50)

(TSA)

Add 1.2mL DMSO to TB for a new kit

62 µl

TSA (tyramide-biotin)

 

PE LAS, Inc (Renaissance) Individual Indirect Tyramide reagent Pack

Perkin Elmer: SAT700B

tyramide-biotin (1:50) = 3.1mL/50 = 62µl

250µl/slide (12 slides) = 3mL + 100µl extra = 3.1mL

3.1 mL

TSA (tyramide-biotin buffer ) (Amplification diluent)

 

12 slides x 250µl = 3mL

3.1 mL

Total volume    

 

Neutravidin-AP (1:500)

18 µl

Neutravidin

 

Pierce:  31002

 

700µl/slide (12 slides) = 8.4mL (make 9mLs); 9mL/500 = 18µl

Add antibody during TSA step

9 mL

1% BR

 

 

Use existing BR in container, already contains tween

-

10% Tween (already in TMN)    
9 mL Total volume    

 

BCIP/NBT

15 mL

TMN

 

 

 

15mL TMN from container, 300µl BCIP/NBT stock solution for 12 slides

Add right before needed

Keep from light

300 µl

BCIP/NBT

 

Roche: 1681 451 (8mL)

15.3 mL

Total volume    

 

4% PFA

10 mL

5X PFA

 

Sigma P6148 (500g)

Aliquot 5X PFA into 5mL portions and freeze at -20ºC

To make just add PBS

Heat solution at 65ºC for 10 min for PFA to go into solution (if necessary).

0.25 µl

10% Tween

 

 

40 mL

PBS

   

50 mL

Total volume    

 

Starting the ISH run

  1. Flush the instrument.  This can be done by pressing the “flush instrument” button in the menu bar of the Gemini program, and clicking “OK” when the description box comes up.  The robot should flush about 50 ml of system liquid.
  1. Wipe the tips with 100% ethanol using a Kimwipe. 
  1. Make sure the two water baths are on.
  1. Fill up the large system liquid water with ultra pure (Milli-Q) the night before a run, allow the liquid to degas naturally.  Do not add system liquid the same day unless the water is degassed, you do not want bubbles in the system liquid which may cause a run to fail.
  1. Empty out the waste containers. 
  1. Open Evoware, select the script you want to run.
  1. Press the start button and the script will begin and ask for the number of slides to be processed. Before entering the slides number and pressing enter, prepare the methanol peroxide solution and place into receptacle.

In Situ-Hybridization Steps

 Prehybridization steps
“Washing” means adding the amount of solution specified in the protocol to all flow-through chambers. “Repeated washing” means multiple cycles of washing. By contrast, “incubation” means that added solutions were left in the chambers for the amount of time specified in the protocol below.  All solutions were provided in suitable containers located on the platform.

  1. Wash 5 times for 5 minutes with MeOH containing 0.7% hydrogen peroxide.
  2. Wash 7 times with 300µL of PBS.
  3. Incubate 2 times for 5 minutes of 0.2N HCl.
  4. Wash 7 times with PBS.
  5. Incubate 2 times for 15 minutes with pre-hybridization solution.
  6. Incubate 1 time for 15 minutes while the temperature is raised to 64°C (no solution is delivered, only a timer is set within the script, this gives the incubator time to warm before probe hybridization).

Hybridization step

  1. The robot automatically delivers the probe to the specified hybridization chambers. Incubate probe at 64°C for 5.5 hours. After 2.5 hours a second aliquot of probe is pippetted. Adding fresh probe halfway through the incubation increases the signal strength of weakly expressed genes.

Post hybridization stringency  washes
Post hybridization washes are carried out at 62°C. Temperature reduction to room temperature is carried out while in cycle 1 of step 1 below:               
Stringency wash solution is prewarmed for 1.5 hours (no longer) on the robot platform in a heated container so that they reach approximately 60°C.  This is necessary to avoid degassing that would occur if room temperature solutions were added to a 62°C hybridization chamber.

  1. Incubate 5 times for 5 minutes with 5x SSC.
  2. Incubate 5 times for 10 minutes with 2x SSC with 50% formamide (Formamide I).
  3. Incubate 5 times for 12 minutes with 1x SSC with 50% formamide (Formamide II).
  4. Incubate 4 times for 8 minutes with 0.1x SSC.

Probe detection reactions
Temperature reduction to room temperature is carried out while in cycle 4 of step 4 in the above stringency washes. All post-hybridization steps are carried out at room temperature.              

  1. Wash 4 times for 5 minutes with NTE
  2. Incubate 6 times for 5 minutes with 20mM iodoacetamide in NTE
  3. Wash 4 times for 5 minutes with NTE
  4. Wash 2 times for 5 minutes with TNT solution
  5. Incubate 6 times for 5 minutes with 4% sheep-serum
  6. Wash 4 times for 5 minutes with  TNT solution
  7. Incubate 2 times for 10 minutes with TNB blocking buffer (PerkinElmer Life sciences, FP1020) 
  8. Wash 2 times for 5 minutes with TNT solution
  9. Wash 2 times for 5 minutes with maleate wash buffer (MWB)
  10. Incubate 2 times for 10 minutes with 1% blocking reagent (Roche)
  11. Wash 2 times for 5 minutes with MWB
  12. Wash 2 times for 5 minutes with TNT solution
  13. Incubate 3 times for 5 minutes with TMN solution
  14. Wash 4 times for 5 minutes with TNT solution
  15. Incubate 4 times for 10 minutes with TNB blocking buffer.
  16. Incubate 2 times for 30 minutes with Anti-DIG-POD in TNB blocking buffer
  17. Incubate 6 times for 5 minutes with TNT solution
  18. Incubate 1 time for 30 minutes with tyramide-biotin diluted with amplification diluent’s buffer (all in TSA kit, PerkinElmer Life sciences SAT700B)
  19. Wash 6 times for 5 minutes with MWB.
  20. Incubate 2 times for 20 minutes neutravidin-alkaline phosphatase conjugate (Pierce 31002) diluted in 1% blocking reagent (Roche 1096176).
  21. Wash 6 times with MWB for 5 minutes.
  22. Wash 4 times for 5 minutes with TNT solution
  23. Wash 2 times for 5 minutes with TMN solution.
  24. Incubate 3 times for 10 minutes with TMN solution containing BCIP/NBT.  This step may be extended depending on signal strength.
  25. Wash 3 times with system liquid water.
  26. Wash once with NTE.
  27. Incubate once for 10 minutes with 4% PFA
  28. Wash 3 times with system liquid water

Disassembly of Hybridization chambers

  1. Remove hybridization chambers from the robotic platform.
  1. Use the clamp remover to remove clamps from assembled hybridization chambers.  Place clamps into a small container to be rinsed.
  1. Place unclamped hybridization chambers face-down into MilliQ water one at a time.  Do not allow chambers with slides to set in water for too long.  Place slide frames into a container to be rinsed later.
  1. Carefully remove slides from water and back plates, let spacers float off and place them, in the metal slide racks.  Take care not to scratch the tissue sections.
  1. Rinse the slide racks briefly in ultra-pure (Milli-Q) water.
  1. Allow the slides to dry in a fume hood at RT for ~10 minutes.  
  1. Coverslip with VectaShield Mounting Medium with Dapi (Vector Laboratories, Inc. cat# H-1200).  (use only 1-3 drops per slide, depending on the number of sections per slide)

Cleaning of Hybridization chambers

  1. Take back plates from the water and place them into containers with ethanol. Leave them overnight.
  1. Take back plates out of ethanol and place them in stainless steel containers, and put them in a dishwasher to be cleaned.
  1. Rinse slide holders and clamps twice in deionized water and leave them out to dry. 
  1. Remove stainless steel containers from dishwasher; place the containers in external stainless steel box and autoclave.

Shut-down

  1. Empty out the waste containers.
  1. Fill up the system liquid container.
  1. Turn off the robot; water-baths can stay on stand-by.


Group Description