I have been studying immunofluorescence technique in rodent forebrain and brainstem sections. I perform cryosectioning (50 um thickness) in sucrose-treated brains (formerly fixed in 4% PFA) and use free floating technique.
I’ve seen several commercial anti-autofluorescense solutions but they are quite expensive.
Hi Osman, you might have some luck in this thread: https://www.researchgate.net/post/How_is_it_possible_to_remove_or_reduce_autofluorescence_from_cryosectioned_arteries
Alternatively: http://wwwfacilities.uhnresearch.ca/wcif/PDF/Autofluorescence.pdf
I remember I used to use ammonium chloride when I was quenching the auto-fluorescence of cerebral organoid slices prior to staining, I believe at 1mM but unfortunately I do not have access to these notes anymore.
Hi Osman,
paraformaldehyde causes itself a significant autofluorescence. If possible, avoid it, when you are doing your next fixation protocol. A useful reference to your problem is
J Histochem Cytochem. 2014 Jun; 62(6): 405–423. doi: 10.1369/0022155414531549PMCID: PMC4174629
Characterizing and Diminishing Autofluorescence in Formalin-fixed Paraffin-embedded Human Respiratory Tissue
A. Sally Davis, Anke Richter, Steven Becker, Jenna E. Moyer, Aline Sandouk, Jeff Skinner, and Jeffery K. Taubenberger
Best regards,
Daniela
Yes, I read about ammonium chloride and alternatively just Glycin. I quite successfully used a blocking buffer containing 0.3 M Glycin + 10% normal donkey serum in PBS to reduce fluorescence background of different kinds in 50 µM free floating sagital sections of mouse brain (though no sucrose treatment). However, increasing with age, I still found autofluorescence to be hassle at ages older than 15 days. In this case, it greatly helps to stay away especially from green dyes (Alexa 488). The best that worked for me was using donkey-Alexa647 at 1:200 dilution. High concentration of secondary made up for poor brightness of 647 and long wavelength circumvented tissue autofluorescence. Hope this helps!
place your slices for 30 minutes in freshly prepared PBS 0.1M+0.05% Na-borohydrate. rinse thoroughly, then incubate for 60minutes in 10%blocking serum.
After the IHC-Reaction and TBS rinse the sections in
destilled water --- short
70% ETOH -- short
1% Sudan- Black-B solved in 70% ETOH -- 10 min
70% ETOH -- short
7% ETOH -- ca. 5 min
Destilled water -- ca. 5 min; mount the sections on slides, air dry them and mount them either in Glycerin-Gelatine (Sigma) or Dianova mounting medium.
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