Dear Histonetters, 

Phil kindly provided a method (with credit to Dr Miller) on blocking
endogenous biotion in tissues using an egg white solution.  Very
interesting and much appreciated from one of our vendor friends, glad to
have them around to help.




From: "Phil Logsdon" <plogsdon@cellmarque.com>
>To: <uvsgc@msu.oscs.montana.edu>
>Subject: quenching endogenous biotin

>I do have some endog. A/B blocking techniques that we have
>used here in our lab for troublesome tissues and I have passed this on to
>other labs who have used them with success.
>This information comes from a workshop taught by Dr. Rodney Miller MD of
>Propath Labs in Dallas, Texas.  The workshop was titled Technical
>Immunohistochemistry and his technique for blocking is as follows...
>This procedure should be performed immediately before application of the
>primary antibody(and  after any protease or HIER steps).  It involves
>incubation of the sections for 20-30 mins with 0.05% free avidin in PBS
>followed by a PBS, then incubation of the sections for 10-20 minutes with
>0.05% free biotin in PBS.  The initial incubation with free avidin will bind
>any biotin in the tissue, and the subsquent incubation with biotin saturates
>all the biotin-binding sites on the avidin.  This effectively blocks any
>subsequent staining of the tissues due to endogenous biotin activity.
>Commercial AB blocking kits are expensive- we dont even sell one!  Dr.
>Miller came up with a very economical alternative that I will relate as
>follows...
>Dr. Miller hypothesized that because commercial avidin is obtained from egg
>whites, perhaps a dilute solution of fresh egg whites could be used.  This
>was indeed the case, odd as it sounds.  Effective blocking of endogenous
>biotin can be achieved by placing the slides in dilute egg white solution( 2
>egg whites diluted to 200ml with distilled water) for 15 mins at room temp.
>Since salt solutions (like PBS buffer) can precipitate out proteins in the
>egg whites, the slides must be thoughly rinsed in distilled water BEFORE and
>AFTER this step.  (This will require some patience for the after part as the
>egg whites are, of course, quite snotty.)  There are dehydrated egg whites
>available that are not nearly as troublesome to work with but they are a bit
>more expensive.  Should you use a product like this a 20% solution works
>well (20 gms/200 ml water).
>Dr. Miller, ever the clever chap, discovered that skim milk is in fact a
>rich source of biotin and uses that for the source of his biotin block.  A
>ten min. incubation in skim milk following the egg white incubation does the
>trick for us.  Sounds a bit goofy, granted, but the results will speak for
>themselves.
>  If this works for you let me know, also, feel free to post this info on
>histonet, but please give the credit to Dr. Miller.
>
>Good Luck!
>Phillip Logsdon
>Cell Marque Corp.
>Austin, Texas
>It is real real hot here.
>
>
>
Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303