RE: Rat BRDU question

From:Chris van der Loos

Dear Nick,
Concerning your double staining problem on dog pancreas combining BrdU and 
insulin IHC:
You didn't mention the use of FFPE sections, but I guess this is the case. 
As I wrote here some time ago you may use just HEIR with citrate pH6.0, 
instead of using the harsh treatment with HCl. Very likely HIER will not 
affect your insulin or glucagon staining, and if you are lucky it is even 
enhancing the staining.
Good luck!
Chris van der Loos
Dept.of Cardiovascular Pathology
Academic Medical Center
Amsterdam, The Netherlands

Nick Madary wrote:
 >Date: 25 Mar 2003 15:30:27 -0600
 >Subject: Rat BRDU question
 >Pancreatic Islets on rat pup pancreas(7 day old pups), we can barely
 >harvest pancreas anyway, and at that age an insulin shows up as little
 >dots if we are lucky, forget an actual Islet. So the pancreas is a
 >growing pancreas because it is from a 7 day old rat pup, the BRDU lights
 >up all over. We want to count the BRDU positive cells in just the
 >Islets(in this case anything that would be insulin positive). So I have
 >tried doing the insulin first then brdu and vice versa using different
 >colored DAB. It is my feeling that the harsh treatment of the HCL and to
 >a lesser extent the sodium borate in the BRDU is destroying any chance of
 >getting the insulins to light up. I tried doing some regular special for
 >pancreatic islets but I still run in to the problem of no real islets at
 >the 7 day mark for a rat.
 >So how can I count BRDU cells in the areas I feel confident is the
 >pancreatic islet only. Also did a glucagon/BRDU doblestain and it did not
 >work either. I do not feel an Alk Phos/HRP is even the issue here.

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