General Research Topics > Neuroscience

brain - autofluorescence

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pandanac:
hi Vgoose and all:

I am new to IF....Could you please shed light on the process of when to use the ammonium chloride to remove or reduce the autofluorescence. I have heard that Sodium borohydrate, Imido black, Tris-Glycine, or Sudan-B can help in countering the effects of auto fluorescence... I am using brain sections perfused with 4% PFA and stored in 4% PFA for 24hrs and then in 30% sucrose..I dont know if this is the correct way of doing it..I am running out of ideas ???.. pleas e help me please  :'(

Koi:
@Pandanac - I recently got the same problem with my brain tissue sections. I think I will try 'Autofluorescence Eliminator Reagent'. I will tell you if it works well.

MikeCU:
I am also getting this exact problem in organotypic brain slice cultures. Koi - any success? Should I do glycine or ammonium chloride BEFORE permeabalization?

Thanks for any comments!

EKaplan:
I am having the same problem with autofluorescence on my cryostat sections, even before any IHC is performed you can see what appear to be fluorescent puncta around cell bodies, under all wavelengths..

Im currently perfusing with 4% pfa, postfixing brains in 4% pfa overnight, then cryopreserving in 30% sucrose in 0.1Phosphate Buffer, then freezing brains in OCT on dry ice.

Anyone figured out the origin of this? With the antibody im using (GAD65) the autofluorescence tends to be brighter than the actual immuno signal.

Suggestions???

thanks

CanuckPhD:
I have found the following article quite good as a resource. It doesn't use brain tissues but the same techniques can be applied. For human Alzheimer's disease brain I usually use the Sudan Black method with decent results.

Control of Autofluorescence of Archival Formaldehyde-fixed, Paraffin-embedded Tissue in Confocal Laser Scanning Microscopy (CLSM). Werner Baschong, Rosmarie Suetterlin and R. Hubert Laeng. J Histochem Cytochem 2001 49: 1565

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