I'm trying to do Immunofluorescence staining for LR-white resin section on formvar coated nickel grids. I have problem in antibodies penetration.
I've tried to use 50mM glycine followed by 0.1% Triton, 10 min each step, but they didn't work. However DAPI is the only thing penetrated and stained the section.
And I would like to inform that :
-Samples are tissue not cells.
-Samples were fixed in 0.5% Glutaraldehyde and 4% paraformaldehyde.
-No osmium tetroxide was used.
- 1ry antibodies were incubated overnight at 4C.
So, any suggestions to come over penetration problem ??
thank you :)
Dear Abeer, as a 'snapshot' - you certainly read already:
Research Article : Triton X‐100 Pretreatment of LR‐white Thin Sections Improves Immunofluorescence Specificity and Intensity by
Salem S. Ghrebi, Gethin Rh. Owen, & Donald M. Brunette
==> Article Triton X-100 Pretreatment of LR-white Thin Sections Improves...
or (NB: before copying and pasting into your browser delete the underline-character in between http: _ and s://....):https_://onlinelibrary.wiley.com/doi/full/10.1002/jemt.20422 (unfortunately for me only PPV)
onlinelibrary.wiley.com/doi/10.1002/jemt.20422/pdf or:
http://scienceservices.de/media/pdf/LR%20White%20Tips%20and%20Tricks.pdf or
Re-evaluation of Epoxy Resin Sections for Light and Electron Microscopic Immunostaining. GROOS et al., JHC, Vol 49, Issue 3, 2001
journals.sagepub.com/doi/full/10.1177/002215540104900313
If you did the technical part already perfectly my first guess would be defect / gammy antibody?
Thank you very much.
I think I have problem with my primary antibodies , because I used them to do IF on paraffin section and there was no signal! the problem is the 2 primaries which I used from different companies !!
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