Re: false positive with HRP and DAB
Thanks to everyone who answered!
Original problem is that I have staining ffpe tissue with HRP-conjugated secondary on my control slide (primary ab left off) Thinking there is a problem with my peroxidase blocking step.
More specifically, My tissue is excised perinatal mouse inner ear, ffpe (4% para x1hr on ice). I cut my sections 4 microns. My primary is an antibody that recognizes GAG epitopes on chodroitan sulfate proteoglycans (but of course it was left off the negative control slides).
I am using the Vectastain elite RTU ABC kit. I deparaffinized and rehydrated these slides, no AR (I ran another batch using cooker with AR solution that dewaxes, rehydrates and peroxidase blocks--no false positive with that control but I am testing the ab with and without AR)then used H202 as stated, then blocked with prediluted NHS is kit. The biotynalated secondary in kit is raised in horse against rabbit and mouse IgG.
Investigators I have read do not report avidin/biotin blocking with inner ear. I am developing in DAB and counterstaining with methyl green.
Some of you have urged me to ditch the bottle of 3% 'supermarket' H202 given me by my PI and get some 30% to dilute fresh each time to 3%. I will dilute in PBS as I fear what methanol may do to the organ of Corti in my sections.
I showed him your emails and he signed the purchase order (growling just a little!)
If any of you know of any particular
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