Hi, 

my protocol was like that below:

1. wash cells with PBS

2. fix them (1-4% formaldehyde or 0,5-1% glutaraldehyde, both in PBS) 

3. PBS

4. add cold 70% Ethanol- 20 min in 4*C

5.PBS

6. 8% BSA in PBS, 1h, RT

7. PBS

8. primary antybodies in 1% BSA, 2h, RT

9. PBSx3

10. secondary antybodies, 1h, RT

11. PBSx3

12. You can use Hoechst or DAPI

I hope that will be usefull for you.

1-     Seed 10k/well in 250 ul

2-     Wash cells with PBS 2x

3-     Incubate cells in 3.7-4% formaldehyde for 10’(dilute from 10% formaldehyde without methanol for even better results)

4-     Fast wash 2x

5-     Permeabilize cells in PBS with 2% Triton X-100 10min at room temperature

6-     Fast wash 2x

7-     Incubate samples 1h in primary antibody with 10% FBS in PBST (Tween20)

8-     Wash 2x 2min in PBST

9-     Incubate 30-60min in secondary antibody 1:500 in 10% FBS in PBST. Add dapi (1:300 from stock )also!

10- Rinse slides in PBST and wash 2x 5min in PBST

11- Mount slips in small drop of mounting media

12-  Cover slips with paper and add even pressure to get rid of extra mounting media

13-  Seal slips with nail polish, dry & visualize

Thank you Selmi and Knobel.

Should I seed in a 48-well or 24-well plate? Because I have no idea how much cells needed for this assay. 

Thanks.

I am seeding cells in 12 well plate, but first i put inside the cover glass (cells are grown on cover glass and then after staining I place them on basic glass and put few drops of fluoromount to make a specimen). Good luck!

You could also utilise protocols for synaptonemal complexes and follow as regular IF protocols.

Hi Arun,

Depending on your imaging platform, I highly recommend FITC-conjugated anti-phospho(Ser139)-H2AX antibody (γ-H2AX-FTIC, Millipore). I have attached my pub with the methodology, and please feel free to contact me if you need additional details.

Article Alkbh8 Regulates Selenocysteine-Protein Expression to Protec...

In order to get clear foci, the primary antibody should be added in the presence of 0.1-0.05%% Triton X-100 (or tween 20) and considerable washings with the same in buffer should be applied after primary and secondary antibodies - more extensive than in usuall immunocytochemistry.

is there anybody who tried the IF assay of gamma H2AX on tissue (testis specifically)