problem with DAB staining

[Mibbi]

Hej at all,

I have a problem concerning DAB staining on spleen cells of mice. I prepared the tissue samples, which were frozen in OCT, in a section cutting mashine and let them air dry for 1 hour. Then I started my staining procedure by putting the slices into ice-cold 100 % acetone to fix them, then another 10 min air dry and after this step the problem occured: When I added 0,3 % H2O2 to block peroxidase, the tissue slices became white and fluffy and were just swimming around in the fluid.

I can t find a reason why this happened! I tried it over and over again, but the same problem occured every time. My supervisor has the suggestion that we should air dry the slices over night and then start the procedure as this is usually done like this. But why should this solve the problem? I mean the slices are very thin (6 micrometers) that it should not take 24h to get them dry!?

May someone here can help me and has a better idea to solve my problem!

thank you all!

cheers,

Michael

[richard03]

Does the H2O2 made in Methanol or water?

[Mibbi]

it s diluted in PBS, first concentration 30 %, then diluted to 0,3 % (1 : 100)

[Cardio]

What type of slides are you using and have you tried another batch of slides (superfrost + or adhesive) to make sure that this isn't the problem?

[richard03]

I agree with Cardio that it is critical to use adhesive or coated slides to preventing sections from falling off.

I also suggest to use methanol for H2O2 dilution so you can avoid white and fluffy air bubbles, especially on frozen sections.

[Mibbi]

thank you for your tips, I ll try so next time when I ve to repeat the staining!