Dear EM experts,

I've been using the following protocol for immunogold labeling for the past couple years with good success.  This morning one of our users has inquired as to why the secondary is incubated at a higher pH than the primary.  I do not know the answer...do any of you???

IMMUNOGOLD STAINING TECHNIQUE 
	Cut sections (preferably processed into acrylic resin) and mount on inert grids such as nickel or gold. 
	Rinse grids in distilled water for 10 minutes. 
	Incubate in pH 7.4 T.B.S. ( tris buffered saline) containing 5% normal serum for 30 minutes. (Serum from same animal as secondary antibody). The concentration of the normal serum may have to be increased to up to 50% to prevent background signal. 
	Incubate in specific primary antibody diluted 1 in 5 with pH 7.4 T.B.S., including 0.1% bovine serum albumin, for 30 minutes. (Check pH after preparation). 
	Wash grids in two changes of pH 7.4 T.B.S. for 5 minutes each, then two changes of pH 8.2 T.B.S. for 5 minutes each. 
	Incubate with immunogold conjugated secondary antibody diluted 1 in 50 with pH 8.2 T.B.S., including 0.8% bovine serum albumin, for 1.5 hours. 
	Wash grids in pH 8.2 T.B.S. for 5 minutes x 2. 
	Post fix grids in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer for 15 minutes. 
	Wash grids in two changes of distilled water for 5 minutes each. 
	Stain grids with uranyl acetate and lead citrate. (If using LR WhiteŽ resin stain in aqueous uranyl acetate.)


Many thanks in advance!!

Danielle Crippen
Morphology and Imaging Core Manager
Buck Institute for Age Research
8001 Redwood Blvd.
Novato, CA 94945
415-209-2046
dcrippen@buckinstitute.org



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