Rat BRDU question

From:

Nick_Madary@hgsi.com

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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