I am studying the structure of cmv in lung tissue using Fluorescent Immunostaining but I get a lot of background autofluorescence (autofluorescence seen on FITC and Tex Red channels). Has anyone performed fluorescent staining on lung tissue and managed to solve this problem?
Hi Christina, the problem is,there are alot of reason which could responsible for autoimmunfluorescence. 1. Take fresh Xylol and Alcohol for dewaxing your sections.
2. The delution medium is a critcal point. Normal serum could made unspecific background staining as well as BSA. 3. TBS or PBS with or without Triton X-100 or Tween. 4. Antigen retrievel increases the background staining. 5. If you are working with fluochrome labelled Streptavidin you need to block the endogenous biotin with acvidin/biotin blocking kit. And 6.of course the anibodies by themself can produce unspecific staining. 7. Finally not to forget the autofluorescence of the tissue. My recommandation: run negative controls under diferent circumstances. Check a dewaxed non staind section under the microskop. And if you do not need a double labeling try to detect your immuno with DAB when all those other thing do not help.
Good luck and best wishes, Ute
Hi Christina,
As I said to other person that ask same question, use NH4Cl 50mM 10 min on your sections before all other treatment on your sample.
You may bleach a part of your autofluorescence due to aldehyde C=0 resonance.
VM
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