Immunostaining no longer working

[debbie_m]

Hi guys!

 

I hope this is the right place to post this!

 

I am posting because over the last three weeks I have failed to get any data from microscopy. My problem is that the BrdU incorporation/immunostaining is not working. I have been doing this protocol for the last 4 months without any problems using U2OS Cell line. Recently, every time I have been takeing the slides I made down to the microscope, the BrdU staining is all over the field of view and it looks the nucleus has "burst" and the DNA is spread all over the coverslip. I repeated it all again today using fresh reagents and this time I couldn't see anything at all. I should also note that I have failed to stain the cells with Dapi on all of these occasions and although I can see a slight blue signal when looking down the microscope, I cannot focus in on a cell!

 

I would really appreciate some feedback/suggestions! If you need anymore information let me know!

 

Thank you in advance for any help!

 

Debbie

[CharonY]

It is hard to tell from the description, but the failure of DAPI is suspicious. First, are you sure the microscope is working? I.e. even without stain you can see your cells well in transmittance light? And second, the cells are Ok?