GAIDO Group Protocols

Two-color FISH Protocol

Steps 1 - 13 based on Andy McMahon's Double Fluorescent Section in situ hybridization. Steps 14 - 38 based on the Gaido lab's bench TSA Fluorescent Section in situ hybridization method. 

Updated Sept. 2008.
Author:  Janan Hensley

Probes were labeled based on the protocol found on the GUDMAP website (GAIDO lab).
Tissue fixation method based on the McMahon lab protocol located on the GUDMAP website.  (PFA to sucrose, frozen sections cut then air dried).  10µm slides air dried for 30 minutes before freezing at -80C.

Hybridization - DAY 1

Note:  Throughout the procedure, try to remove as much previous solution as possible before going into a new solution; however, DO NOT let the tissue completely dry out.

1.  Air dry previously frozen slides under an air-flow hood for 15 minutes.
(Step 1 has been optimized by the Gaido lab.)

2.  Fix sections in 4% PFA/PBS for 10 min.  Do not discard, save for step 6.

3.  Rinse in DEPC PBS, 3 x 5 min each.

4.  Treat with 10µg/ml of Proteinase K in PBS for 10 min.
Be careful as to the strength of the PK as it varies from lot to lot – this timing/concentration should be adjusted every time a new stock is made.

5.  Rinse quickly in DEPC PBS.

6.  Fix in 4% PFA/PBS for 5 min.

7.  Rinse in DEPC PBS, 3x 5 in each.

8.  De-acetylation:  In a glass histology jar on a stirrer combine:

DEPC H2O 200ml
Triethanolamine  2.66ml
37% HCl 0.35ml
   
Begin stirring and add:  
*Acetic anhydride    0.75ml

Place rack of slides in jar while stirring, stir for 10 min.
*Add 375ul of acetic anhydride, wait 5 min, then add the second 375ul.
Acetic anhydride is only good for 5 minutes, adding another aliquot is necessary.

9.  Rinse in DEPC PBS, 2x 3 min each.

10. Rinse with 0.85% NaCl for 3 min.

11.  Wash with 70%EtOH/0.85%NaCl for 5 min.

12. Wash with 95% EtOH for 5 min.

13.  Air dry for 10 min.

14.  Hybridization:  Dilute probes with hyb buffer such that the probe concentration is between 300ng/ml to 500ng/ml.

Heat the diluted probe to 80ºC for 30 seconds, then heat to 65ºC for 10 min, cool to room temp.  Arrange slides horizontally in a humidified  chamber (PBS), remove excess solution, apply ~150µl of hybridization buffer with probe to each slide, and apply parafilm.
Incubate at 55º - 58º C O/N.

Note:  typically use 300ng/ml for strong probes, 400ng/ml for medium probes and 500ng/ml for weak probes. 

15.            Hyb - added 150µl of probe to each slide, cover with parafilm, incubated O/N at 55ºC.  (Make humidified chamber for o/n incubation with PBS.)

DAY 2

NOTE:  keep reagents at 55 - 58ºC during incubation.
16.  5x SSC - 55ºC - 25 min

17.  Formamide I - 55ºC - 50 min

18.  Formamide II - 55ºC - 1 hour

19.  0.1x SSC - 55ºC - 25 min

20.  TN wash 3x 5 min each, RT with agitation

21.  1% H2O2 in TN - 20 min (no Tween)

22.  TN wash 3x 5 min each, RT with agitation

23.  Block (PE) in TNB - RT - 30 min

24. ** αDig POD or anti-DNP HRP (1:500) - RT - 1 hour

25.  TNT wash - 3x - 5 min each - RT with agitation

26. ** Apply ~100µl TSA Cy3 or TSA fluorescein (1:50) - 15 min incubation (use humidified chamber (PBS))

27.  TNT wash 3x 5 min each - RT with agitation

28.  TN wash 3x 5 min each, RT with agitation (gets rid of Tween)           

29.  1% H2O2 in TN - 30 min (no Tween) - optimized by the Gaido lab

30.  TN wash 3x 5 min each, RT with agitation

31.  Wash with TNT, 1x 1 min (just enough to add back some Tween)

32.  ** αDig POD or anti-DNP HRP (1:500) - RT - 1 hour (use humidified chamber (PBS))

33.  TNT wash 3x 5 min each - RT with agitation

34.  ** TSA-Cy3 (1:50) or TSA-Fluorescein (1:50) - 15 min incubation (use humidified chamber (PBS))

35.  TNT wash 3x 5 min each - RT with agitation

36.  H2O rinse

37.  Coverslip once dry & use VectaShield Mounting media for flourescence with Dapi (Vector Laboratories, Cat# H-1200, 10ml)

Reagents

Volumes can be adjusted for the type of container used.

4% PFA/PBS           

Proteinase K (10ug/ml) in PBS

De-Acetylation:

DEPC H2O 200ml
Triethanolamine  2.66ml
37% HCl 0.35ml
on a stirrer add:  
*Acetic anhydride    0.75ml

after 5 min add another acetic anhy.
*acetic anhydride is only good for 5 minutes, add another 0.375ml after 5 minutes.

 

0.85% NaCl (in DEPC H2O)

70% EtOH/0.85% NaCl (in DEPC H2O)

95% EtOH (in DEPC H2O)

Hybridization buffer
Ambion hybridization buffer - use 1ml per probe
heat to 65ºC for 10 minutes, cool to RT
15µl 100mg/ml DTT
15µl 10mg/ml tRNA

5X SSC            preheat to 65ºC for 10 minutes before use.
37.25ml            DEPC H2O
12.5ml            20x SSC
250µl            10% Tween 20
50ml            Total volume

0.1x SSC            preheat to 65ºC for 10 minutes before use.
49.5ml            DEPC H2O
250µl            20x SSC
250µl            10% Tween 20
50ml            Total volume

Formamide I            preheat to 65ºC for 10 minutes before use.
14ml            DEPC H2O
3.5ml            20X SSC
175µl            10% Tween 20
17.5ml            formamide
35ml            Total volume

Formamide II  preheat to 65ºC for 10 minutes before use.
15.75ml       DEPC H2O
1.75ml         20X SSC
175µl          10% Tween 20
17.5ml        formamide
35ml           Total volume

TNB            preheat to 65ºC for 10 minutes before use.
5ml              10X TN
250µl           10% Tween 20
45ml            DEPC H2O
0.25g           blocking reagent*
50ml            Total volume
*Add blocking reagent to solution then heat so that it goes into solution.

1x TN           
250ml              10x TN
2250ml            DEPC H2O
2500ml            Total volume

1% H2O2 (x2)
Make 2 different solutions to accommodate the 2 steps.
500µl            H2O2            Do not reuse the same 1% H2O2 for the second step.
50ml             1x TN
50.5ml          Total volume

Anti-digoxigenin-POD            1:500 in TNB

Anti-DNP HRP            1:500 in TNB

1X TNT
125ml              10x TN
6.25ml             10% Tween 20
1118.75ml       DEPC H2O
1250ml            Total volume

Tyramide-Cy3 (1:50)
- followed instructions in kit for diluting, incubated for 15 minutes.

Tyramide-Fluorescein (1:50)            followed instructions in kit for diluting, incubated for 15 minutes.

10x TN
For 1L add the following:
Tris 121.5g
NaCl 87.95g
Add ultra pure water up to a volume of 800ml and stir until solid components dissolve. Adjust pH to 7.5 and add more ultra pure water up to a final volume up to 1L.
Store at RT.

10% Tween 20 - dilute Tween 20 to 10% with DEPC H2O

** The label of the strongest probe should be added first.  Example:  the strongest probe is labeled with DIG, the Anti-Dig-POD (step 24) should be added first then TSA-Cy3 (step 26).  The medium or weak probe labeled with DNP, the anti-DNP-HRP should be added second (step 32) followed by TSA-fluorescein (step 34). If the strongest probe was labeled with DNP then add the anti-DNP first followed by TSA-Fluorescein then add the anti-dig for step 32 and TSA-Cy3 for step 34. Optimized by the Gaido lab.        

Materials list


Chemical

company

catalog #

DEPC

Sigma

D5758

Triethanolamine

Sigma

90279

37% HCl

Sigma

258148

Acetic Anhydride

Sigma

A6404

PF

EMS (Electron Microscopy Sciences)

19208

PK

Roche

3115836001

5M NaCl

Ambion (ABI)

AM9759

PBS

Sigma

P3813-10Pak

Ethanol

Sigma

E7023

Hybridization buffer

 Ambion (ABI)

B8807G

DTT

Sigma

43815

yeast tRNA

Ambion (ABI)

AM7119

20x SSC

Ambion (ABI)

AM9763

Tween 20

Sigma

P1379

Formamide

ISC Bioexpress

606

Blocking reagent

Perkin Elmer

FB1012

anti-DIG POD

Roche

11207733910

anti-DNP HRP*

Perkin Elmer

NEL747A001KT

TSA-Cy3

Perkin Elmer

NEL744B001KT

TSA-fluorescein

Perkin Elmer

NEL741001KT

Tris

Sigma

T1503

NaCl

Sigma

S3014

Superfrost Plus Slides

Fisher

12-550-15

H2O2

Sigma

216763

DMSO

Sigma

D8418

Filter Paper (Whatman)

Sigma

Z240109

Parafilm

Sigma

P7543

Coverslips

Corning

12531C (2935-224)

Dapi mounting media

Vector Labs, Inc

H-1200

*TSA plus DNP (HRP) System kit (only need the anti-DNP HRP conjugate from kit but have to buy whole kit.)