Molecular Cytogenetics/Molecular Pathology Laboratory

FFPE FISH Protocol

Materials Required
Anti-digoxigenin Fluorescein: Roche (11207741910)
Alexa 594: Invitrogen (S-32356)
CAS Block: Invitrogen (008120)
Goat Serum: Sigma (G9023-10ML)
DAPI Antifade: Invitrogen (P36931)
Tween 20: Sigma (P9416-100ML)
Xylenes: Fisher (X3P-1GAL)
70% Ethanol: Fisher (HC1000-1G)
95% Ethanol: Fisher (HC-1100-1G)
100% Ethanol: Fisher (23-794206)
37% HCl: Sigma (258148-500ML)
20XSSC: Invitrogen (15557-036)
Fungal Proteinase K: Invitrogen (25530-15)
Formamide: Invitrogen (15515-026)
Target Retrieval Solution (pH 6.1): Dako (S1699)
Day: 1
1. Cut 5 um sections on salinized slides.
2. Put sections in incubator at 56 C overnight.
Day: 2
1. Take slides from incubator and de-parafinize slides in xylene 3 times for 5 minutes each.
2. Hydrate with ethanol treatment: slides in 100%, 90%, and 70% for 2 min each step.
3. Rinse in distilled water for 5 min.
4. Put slides in 0.2 N HCl for 10 min
5. Rinse in distilled water for 4 min (at this step, should put enzyme into water -see step 10).
6. Rinse 2x SSC/ Tween 20 (50 ml+ 7 drops) @ RT for 5 min.
7. 2x SSC @ 80º C (no tween) for 10 min.
8. Rinse in distilled water for 3 min.
9. Boil slides in 1X Target Retrieval Solution 3 min ( modified 0.5M Sodium Citrate buffer)
10. Rinse 2x SSC/ Tween 20 (50 ml+ 7 drops) @ RT for 5 min.
11. Enzyme (Proteinase-K*) - (400µl enzyme+40 ml 2xSSC) at 55º C for 5 min.
12. Rinse with 2x SSC/ Tween 20 (50 ml+ 7 drops) @ RT for 5 min.
13. Dehydrate with Ethanol 70%, 90%, 100% for 2 min each.
14. Air dry
15. 70% Formamide @ pH 7.0 @ 73º C for 5 min.
16. Cold ethanol dehydration (ethanol put in -80ºC for at least 2 hours) in 70%, 90%, 100% for 2 min each.
17. Air dry.
18. Denature probe @ 73º C for 5 min.( put probe tube in water bath)
 19. Immediately transfer probe tube on ice for 5 min.
20. Add probe 20 µl to each slide.
21. Place rubber cement around cover slip.
22. Heat slides on moat @ 94º C for 5 min.
23. Transfer slides in humidity chamber and put in incubator @ 37ºC for overnight.
Day: 3
1. Remove rubber cement and,
2. Rinse slides in 2x SSC/ Tween 20 @ 45º C for 2 min.
3. Rinse 2x SSC/ Tween 20 @ RT 3 times for 2 min each.
4. Put slides on a flat surface and cover with CAS block (10 % goat serum plus 90% CAS block) RT for 10 min.
5. Remove CAS block by bloating on paper towel and put 400 µl of antibodies on each slides for 1 hour, (to
prepare antibodies see below).
6. Rinse in 2x SSC/ Tween 20 @ RT 3 times for 2 min each.
7. Add 10 µl of DAPI on each slides, cover with glass-slip.

TECHNICAL NOTES

0.2N HCl: To make 1000 ml of 0.2 N HCl; add 16 ml of 37% HCl in 984 ml of ddH2O.
Proteinase-K Treatment: Must work up the optimized time and temperature for the enzyme treatment. Literature described
optimized temperature for the Proteinase-K is 55ºC. Also initial time for treatment may be tried at 5 min initially and
gradually increased or decreased according to results of hybridization.
Initially suggested Proteinase-K treatment temp/time is 37ºC for 10 minutes. If weak or no signal are seen then citrate
treatment may be added first and then after seeing the results enzyme time can be increased. Overtreatment would result
in digestion of cells and hollow honey-comb like appearance of cells without signals. In this case reduce the enzyme time.
Citrate treatment: Insufficiently hybridized sections or sections with weak signals can be treated with citrate. We have used
Target Retrieval Solution (pH 6.0) with good results in certain cases. Citrate treatment can be used in microwave or
pressure cooker. Slides are put in 1X Target Retrieval Solution and container is placed in water container (preferably
1000ml beaker) and is heated for 3 minutes. Slides are washed in 2XSSC/Tween20 afterwards for 5 min. This step is
added before Proteinase-K treatment (step 11) and remaining protocol should follow as above. Citrate time may be
adjusted according to the results, and can be increased if tissue is preserved but has no signals.
70% Formamide: To prepare 50 ml of 70% Formamide. Add 5 ml of 2xSSC+ 10 ml of ddH2O+ 35 ml of Formamide and 55
µl of 37% HCl. Keep it at 73ºC.
CAS block: On day three prepare approximately 1 ml of CAS block for each slide. Add 10% goat serum with 90% CAS
block. Take 400 µl of block to cover slides, in the step 4 on day 3.
Antibody preparation: Anti-digoxigenin-Fluorescein and Streptavidin-Alexa Flour 594 each is added to the previously
prepared CAS block in the concentration of 1:500 (for 1 ml add 2 µl of Anti-dig and 2 µl of Alexa 594). Slides are treated
with this step for 1 hour.

Nallasivam Palanisamy
MCTP