RE: [Histonet] Background Staining

From:"Bonnie Whitaker"


When I was a rep for an immuno vendor, I did technical training on the cap
gap method, anyway, we frequently saw the phenomenon you describe of
staining on blank slides that we occasionally used to pair with a
tissue-bearing slide to form the cap gap.  I think it's a function of a
strong charge.  BTW, we didn't see this as much on slides that underwent
HIER or digestion, but that doesn't help your FS protocol.

Bonnie Whitaker
-----Original Message-----
[] On Behalf Of Margaret
Sent: Wednesday, May 19, 2004 9:58 AM
To: 'Smith, Emily';
Subject: RE: [Histonet] Background Staining

Dear Emily,

That's a strange one. Have you titrated your secondary antibody
sufficiently? what blocking solution do you use? Have the slides
inadvertently dried out at any stage of the procedure? 

These are a few thoughts that spring to mind. The other thing that I would
look at would be to try charged slides from another supplier, or try
Polysine or APES slides for comparison on indeed another batch of slides. 
That's my 2 penny worth - maybe someone else will have something more
helpful to say and no doubt you have thought of some or all of these ideas

Good luck.



Margaret Blount
Chief Technician
Clinical Biochemistry
University of Cambridge
Addenbrooke's Hospital
Hills Road
CB2 2QR 
-----Original Message-----
[]On Behalf Of Smith,
Sent: Wednesday, May 19, 2004 3:42 PM
Subject: [Histonet] Background Staining

Hello All,
In the course of a recent experiment, I saw strong background staining
on charged slides with frozen cells in OCT (optimal cutting temperature)
when treated with streptavidin-HRP. 
The staining appears over the entire glass surface not only where the
cells and OCT were.  This all-over-staining also appears on clean
(without OCT on them) slides that are processed with the OCT slides.  I
have tried extensive water washes and the results remain the same.  No
primary antibody is added and the results are the same.
Have you seen this before? Do you have any suggestions? 
Thank you for your help.

Emily A. Smith
CuraGen Corporation

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