I have a set of substrates which I prepared from collagen, some of which were chemically crosslinked (genepin), and subsequently seeded with cells for a dosage experiment. I later stained for Ki67 and counterstained DAPI for confocal fluorescence imaging.
The samples which were crosslinked exhibit unreasonably high background in the red channel (Cy5). The background is so strong that when raising the exposure time to what I would need to see the Ki67 antigen, it is completely masked by the background. What can be causing such a high background? How can I better visualize Ki67 antigen? I only seem to be having an issue with the crosslinked collagen
Note*
primary antibody - rabbit derived
secondary (Cy5 conjugated) - goat
collagen (type 1) - bovine
4% formaldehyde fixation, BSA used as universal blocker, blocking buffer contained 10% goat serum
I am wondering if there could possibly be some undesirable reaction with the Collagen or BSA (if contaminated with IgG) and the secondary antibody since goat and cow are related. But I don't think this is it, as I am not having this issue with the non-crosslinked substrates
You would better investigate the adhesive medium in the slides, or the type of your visualizing system, if it is enzymatic it is preferred to block the endogenous enzymes
Did you tested autofluorescence of collagen substrate without secondary (Cy5 conjugated) - goat Ab? Maybe level is high due to crosslinking.
As one more tip to prevent unwanted residual formaldehyde activity is ammonia in blocking media.
Daniil,
Yes actually, shortly after opening this thread I checked autofluorescence of substrates and indeed they did have a high level due to crosslinking (in the Cy5 channel only)....Next time I do this I will use Alexa 488 conjugated secondary Ab
Also I will try a cold Methanol fixation in effort to alleviate another issue I was experiencing. I was seeing a lot of expression in the membrane and visualizing a lot of morphological characteristics of the cell/membrane. It was making it difficult to discern a Ki67 positive cell vs. negative. My PI suggested I try a longer permeabilzation to ensure the antibodies were not getting 'trapped' in the membrane. I think a methanol fixation will take care of the whole membrane issue all together
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