[Histonet] NaCl in antibody diluent

[Robert Chiovetti]

Jennifer,

I can't speak to IHC, but I've done quite a bit of immunolabeling for electron microscopy.  We routinely used NaCl at 0.9% (by weight) in diluents and washes with pretty good success.  That's considered to be "normal saline," a physiologically relevant concentration.  0.9% by weight is 150 mM, if I recall correctly.

We also used to use *very* high NaCl concentrations in our washes when we had background problems, and the high salt would frequently get rid of the background staining.  In those cases we'd use about 5X normal saline (4.5% NaCl by weight) as the rinse after the primary and secondary Ab's.  You just have to remember to return to 0.9% normal saline for a couple of washes before continuing with the labeling procedure.

Don't ask me to explain why this worked, but it frequently cleaned up messy background problems.  A biochemist friend of mine says this is similar to what he does when he cleans and preps his gel columns after using them.  He uses high salt to remove traces of proteins and contaminants from the columns.  The way he explains it, the high salt makes most proteins completely collapse so they're no longer "sticky" and they can't bind to anything.  The strange thing is, the specific Ab-Ag binding seemed to survive the high salt just fine.

And my disclaimer: This frequently (but not always) worked for immunoEM.  I have no experience with IHC, but it might be worth a try...

Cheers,

Bob
 
Robert (Bob) Chiovetti, Ph.D.
Southwest Precision Instruments
The Desert Southwest's Microscopy Resource
www.swpinet.com
132 North Elster Drive
Tucson, AZ 85710-3212
Tel./Fax 520-546-4986
Member, Arizona Small Business Association
(www.asba.com)

----- Original Message ----

Has anyone tried NaCl in there diluent buffer for IHC, if so what
concentration. I read about it in DAKO's immunostaining methods booklet
but they don't state the concentration. This is my last chance with my
background problem. I have a sticky collagen issue I cant get rid of.






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