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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.

2. Wipe the tips with 100% ethanol using a Kimwipe. 

3. Make sure the two water baths are on.

4. 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.

5. Empty out the waste containers. 

6. Open Evoware, select the script you want to run.

7. 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.

2. Use the clamp remover to remove clamps from assembled hybridization chambers.  Place clamps into a small container to be rinsed.

3. 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.

4. 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.

5. Rinse the slide racks briefly in ultra-pure (Milli-Q) water.

6. Allow the slides to dry in a fume hood at RT for ~10 minutes.  

7. 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.

2. Take back plates out of ethanol and place them in stainless steel containers, and put them in a dishwasher to be cleaned.

3. Rinse slide holders and clamps twice in deionized water and leave them out to dry. 

4. Remove stainless steel containers from dishwasher; place the containers in external stainless steel box and autoclave.

Shut-down

1. Empty out the waste containers.

2. Fill up the system liquid container.

3. Turn off the robot; water-baths can stay on stand-by.

Group Description

 

 

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