Sender: Confocal Microscopy List CONFOCAL@LISTSERV.ACSU.BUFFALO.EDU
From: Mario Moronne <mariomm@MH1.LBL.GOV>
Subject: anti auto fluorescence

Standard formula found in various sources:

Put sample in freshly made NaBH4 1 mg/ml in pH 8.0 PBS for 15 minutes.

Bubbles of hydrogen gas should slowly appear. If you don't see any bubbles you probably need new NaBH4. Buy a new bottle of the latter and keep it absolutely desiccated. Freshly made means take dry powder and mix with buffer. NO STOCK SOLUTIONS! This material has a high potential for explosion and is very caustic.

I find that it works quite well when used as described. An alternative is cyanoborohydride. Does the same thing but is gentler. If I remember correctly it does generate cyanide as a byproduct. So, pick your poison.

The use of these reagents is particularly suited to reduce the reversible Schiff's bases that are formed by the aldehyde-NH2 reaction and lead to autofluorescence, especially when using glutaraldehyde. If you can use paraformaldehyde for fixation, the reduction step is often unnecessary and autofluorescence is low.

Remember, if it doesn't bubble, you may be in trouble.

Regards,

Hi,

Does anyone have the basic protocal for sodium borohydride(sp?) anti-auto fluorescence wash. Thanks.

Jonathan

-- Jonathan T. Boyd Fox Chase Cancer Center 7701 Burholme Ave. Philadelphia, PA 19111 jt_boyd@fccc.edu

Mario M. Moronne, Ph.D. NanoMed Technologies 1561 Posen Ave Berkeley, CA 94706 drmario@nanomedtech.com