DAB and beta-gal again
Sorry to bother you again, but could anyone help?
To introduce myself, I am a researcher used to work on cardiovascular
physiology, performing anaesthesia, surgery...and I am not familiar with
histology. I have been working for one year on cardiac tissue morphology,
and I should follow a training course soon to start beeing more
professional than I am in histo techniques and analyses.
I have a couple of slides with cardiac tissue full of adenovirus particles
loading with lacZ and expressing beta-galactosidase activity. On the
control slide, with no primary antibody targeted to macrophages, I loaded
with a biotinylated secondary antibody, followed by avidin-HRPO and DAB on
a Ventana program. Maybe it's totally not surprising, but I obtained the
characteristic brown color. Is it normal or just an artifact?
I would be grateful if I can even receive a tiny comment!
All the best
I need your experience as I am totally discovering your world HISTOLOGY.
In immunoassays, I know that the most common substrate for
beta-galactosidase is BCIG (bromochloroindolyl-beta-D-galactopyranoside).
However, I recently did a control slide with tissue I knew there was
endogenous beta-gal activity.
Steps were the following:
- No primary antibody
- Biotinylated secondary antibody
I obtained a brownish color where I know there is beta-gal protein. Is it
Any comment of advice will be very greatly appreciated.
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