GAIDO Group Protocols
One-color FISH Protocol.
Steps 1 - 13 based on Andy McMahon's Double Fluorescent Section in situ hybridization. Steps 14 - 26 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 -80ºC.
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, incubate for 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.
Hybridization: Dilute probes with hyb buffer such that the probe concentration is between 300ng/ml to 500ng/ml. (Use Ambion’s Hybridization Buffer)
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.
14. 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.
15. 5x SSC - 55ºC - 25 min
16. Formamide I - 55ºC - 50 min
17. Formamide II - 55ºC - 1 hour
18. 0.1x SSC - 55ºC - 25 min
19. Block (PE) in TNB - RT - 30 min
20. αDig POD (1:500) - RT - 1 hour, use humidified chamber (PBS)
21. TNT wash 3x 5 min each - RT with agitation
22. Apply ~100µl TSA Cy3 - 15 min incubation, use humidified chamber (PBS)
23. TNT wash 3x 5 min each - RT with agitation
24. H2O rinse
25. 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.
All H2O additions use DEPC treated H2O.
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.
Anti-digoxigenin-POD - 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.
10% Tween 20 - dilute Tween 20 to 10% with DEPC H2O
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.
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 |
TSA-Cy3 |
Perkin Elmer |
NEL744B001KT |
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 |
