[Histonet] PGP9.5 staining -- how to get rid of hair follicle background staining?

[Margaret Blount]

Hair fibres tend to be very "sticky"; the follicles, ie, inner and outer
root sheaths don't, until you get to the level at which the cells are
cornified. Also endogenous biotin will be found in the sebaceous gland in my
experience, I never had any problems with it in the follicle itself.

All I can suggest is wash well in PBS/TBS and increase your blocking to 10%
(or maybe more) normal serum from secondary antibody host prior to the
primary. I always used TBS for staining skin and hair follicles and I
believe it is supposed to give a cleaner result than PBS. Have you titrated
your antibodies sufficiently? If you still get staining in the hair fibre,
then I think you may have to live with that, but you shouldn't get
background on the follicle cells. 

I hope this helps, good luck.

Margaret

Margaret Blount
Chief Technician
Clinical Biochemistry
University of Cambridge
Addenbrooke's Hospital
Hills Road
Cambridge
CB2 2QR 


-----Original Message-----
From: Edmondson David (RBV) NHS Christie Tr
[mailto:David.Edmondson <@t> christie-tr.nwest.nhs.uk]
Sent: Friday, July 23, 2004 2:56 PM
To: 'Cao, Xudong'
Cc: Histonet (E-mail 2)
Subject: RE: [Histonet] PGP9.5 staining -- how to get rid of hair
follicle background staining?


Have you tried applying a biotin blocking kit in the procedure?
Dave
Christie, Manchester

-----Original Message-----
From: Cao, Xudong [mailto:Xudong_Cao <@t> Brown.edu]
Sent: 23 July 2004 14:02
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] PGP9.5 staining -- how to get rid of hair follicle
background staining?


Dear all,
 
I am trying to stain for nerve fibers innervating the skin samples that I
have. While I am lucky to get the nerve fiber singnals, I am also getting
overwheelingly strong background staining from the hair follicles. I know
the follicle stains are background since they are also stained in both of my
controls where primary and secondary are omitted  are also stained. My
question is: how to get rid of the hair follicle stains? any suggestions? I
am using Vector's ABC and SG substrate. Thanks
 
Xudong Cao, Ph.D.
Biomedical Center
Brown University
 

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