[Histonet] hematoxylin and DAB/Alkaline Phosphatase staining

[Cheuk Ho Tam]

Dear Histonet Members,

I am currently staining 20 um cryostat sections of brain tissue and visualizing with DAB along with hematoxylin counterstain.  For sections stained with the same concentration of primary antibody, I seem to get lighter DAB staining for slides with hematoxylin than ones without.  Is it possible that hematoxylin can interfere with DAB staining?  I am using the ABC kit from for DAB and pre-made Hematoxylin QS, both from Vector Labs.

On a different note, I've noticed some sponginess in occasional sets of slides.  I'm wondering what can be the cause.  A labmate told me that it's probably something wrong with the fixation or cryoprotection.  I've treated the brain tissue the same for the batch, and only one slide set had sponginess.  I fix in 4% PFA for 24 hours, cryoprotect with 30% sucrose for 1-5 days, and mount in OCT.  Any suggestions would be appreciated.  

Thank you for your help.

Sincerely,
Cheuk