I wonder to know if immunostaining using DAB is sensitive to amount of tissues as il use drosophila embryo i cannot know exact amount of tissues and for some mutant i have very few embryo. Is this could influence staining intensity ?
Many thanks for your help.
Hi Dufourt
Like any other staining DAB is also a sensitive method, if u worried about the staining intensity based on the sample size, i will suggest you the antibody coated with HRP polymers this will help to amplify the signals like your case.
I hope the following paper will help you some.
http://www.ncbi.nlm.nih.gov/pubmed/23351717
all the best.
Thanks a lot for this answer but my question is more précise about antibody amount compare to sample amount. For example in some mutant some people claim that some protein are overexpress even if thé mutant give less sample . Is it real or could we titrate thé antibody?
Not an easy question i thought.
DAB can be reduced by other substances, e.g. catalases. Next there exists a penetration front for DAB into both fixed and unfixed tissue. I worked in my scientific youth with Callifora erythrocephala, insect tissue will reduce DAB. Thus: quantification is not possible, antibody amount to sample amount is I think not possible, if DAB is involved for quantification. By the way an analogous problem exists with fluorescence. Hope I understood and answered your question well.
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