I have been attempting to analyse collagen expression in cells that have
been transfected with an alpha2(I) collagen expression construct, to examine
the localisation of the exogenous protein.
To do this, I have used a Seralab goat anti-type I collagen antibody, in
combination with FITC-conjugated rabbit anti-goat but have not met with
much success - even examining 3T3 cells. A big problem has been with background
fluorescence. I've also read that ECM proteins are sensitive to a number of
fixatives. Is 4% paraformaldehye OK for this application? Finally, are there
any growth conditions that optimise the deposition of the matrix? Would cell
density be a factor in visualising matrix proteins?
Any tips / foolproof protocols would be most welcome
Thanks in advance
Neil French (grgnsf at picr.cr.man.ac.uk)