FISH + Immunostaining protocol

1. Add 2ul (1:500 stock) EDTA to 1 mL formaldehyde.

2. Spray dissecting dish w/ EtOH and wipe. Fill well with PBS.

3. Fix dissected testes in 500ul formaldehyde+EDTA for 30 min on rotator.

4. Wash 2x w/ PBST+1mM EDTA (1:500) for 10 min.

5. Wash final time w/ PBST+1mM EDTA for 30 min.

6. Prepare 100 ul 1° antibodies in 3% BSA in PBS+1mM EDTA (1:500 stock)

Incubate 4 hr @ RT or overnight @ 4°C

7. Wash 2x with PBST+1mM EDTA for 10 min.

8. Wash final time w/ PBST+1mM EDTA for 30 min.

9. Prepare 100 ul 2° antibodies in 3% BSA in PBS+EDTA (1:500)

All 2° abs at 1:200 conc

Overnight at 4°C

10. Wash 3x w/ PBST+1mM EDTA for 10 min at RT, keeping light exposure at a minimum from here on out.

11. Fix again with 500ul Formaldehyde+EDTA for 10 min.

12. Wash w/ PBST+EDTA for 30 min.

13. Rinse w/ PBST (NO EDTA)

14. Add 100ul RNase A solution (2 mg/ml in PBST) at 37° for 10 min.

15. Wash w/ PBST+EDTA

16. Rinse w/ 2x SSCT (2x SSC with 0.2% of Tween-20) +EDTA

17. Wash w/ 100ul 2xSSC + 20% formamide (80ul SSC: 20ul formamide) for 10 min

“” 50% formamide “” for 10 min

18. Hybridization:

50 µl formamide

10 µl 20x SSCT

2 µl 50 mM EDTA

20 ul 50% dextran sulfate (Don’t use more than 1 year old one)

15 ul probe (e.g. 7.5 µl of Cy3 probe and 7.5 µl of Cy5 probe)

3 µl dH₂O

2 min @ 91°C [in heat block with water!]

Then overnight @ 37°C.

19. Wash 3x with 2x SSCT+EDTA for 20 min.

20. Add 1 drop DAPI (+mounting medium).