[Histonet] hematoxylin and DAB/Alkaline Phosphatase staining
Cheuk Ho Tam
ctam <@t> stanford.edu
Wed May 11 19:09:17 CDT 2011
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.
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