[Histonet] staining of negative controls with IHC

[Rene J Buesa]

There are many who prefer the pressure cooker because of the low incubation period but I personally did not like it because of background issues as you describe and because some tissues peel-off the slides (too much heat).
  Because of that I ended using a HIER step in a buffer (citrate or EDTA depending on the pH) initially heated to boliling temperature in a microwave oven and the actual HIER to take place in a steamer during 20 minutes + 20 minutes out of the steamer. All problems were resolved this way. Give it a try.
  René J.

MaryAnn Dixon <DixonM <@t> vetmed.ufl.edu> wrote:
  We are currently retrieving animal tissue in a pressure cooker with a program of 125 degrees for 30 secs. This protocol was taken from biocare but I'm sure this is for human tissue. Most of the tissue is retrieved with Biocare's Reveal (pH 6.0). We are receiving some background staining of our negative controls whereas if we did not retrieve them, there is none. We were staining for KI-67 which is a nuclear stain. I am using a tonsil for the control. The negative control showed some sporatic cytoplasmic staining on the Universal negative control from biocare as well as deionized water. Since then we have taken the temperature down to 115 degrees and increased the time to 25 minutes. It seems to have decreased the cytoplasmic staining but there is still some lingering. Anyone out there have any ideas??? I'm sure there is a better protocol for retrieving animal tissues. It has to be a time and temperature thing!


MaryAnn Dixon
Biological Scientist
Anatomic Pathology
University of Florida
School of Veterinary Medicine
352-392-2235 ext 4517

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