Re: immunostaining NMDA receptor proteins

From:Phillip Huff

Hello Mary,
I have had success with the following antigen retrieval techniques in amplifying a transmembrane protein expressed in connective tissues of muscle tissue:

(1) Microwaving in Coplin Jars in 0.05 M glycine-HCl (pH 3.5), 2x, 5 min each, changing the solution after each 5 min microwave step

(2) Microwaving in Coplin Jars in 0.8 M urea 2x, 5 min each, changing the solution after each 5 min microwave step

I also fix in PLP (2% paraformaldehyde, 0.1 M L-lysine, 0.1 M sodium meta-periodate in 0.1 M Na2HPO4 (pH 7.4)) solution (made immediately prior to use, important) for 20 min at 4˚C and rinsed three times for five min in 1x PBS. This seems the maintain the antigenicity of my target better.

In addition, I also perform a permeabolization step with acetone for 20 min at -20°C after antigen retrieval. Nothing else seems to work for me.

Hope this helps


Mary B. Olive wrote:
Hello, all.  Does anyone have any suggestions or tips for immunostaining NMDA receptor subunit (and associated) proteins in 4% parafomaldehyde fixed rat cortex?  We currently use the Vectastain HRP kits (Vector Labs) with DAB as a chromogen.  I am especially interested in hearing about your experiences with different forms of antigen retrieval (esp. autoclave) or fixation techniques/times that may improve the signal.
Thanks in advance,
Mary B. Olive
Research Assistant
Pediatric Neurology
Children's Hospital of Michigan
Wayne State University

Phillip Huff
M. Sc. Student
Office: B876

Chemistry and Biochemistry Department
4401 University Drive
University of Lethbridge
Lethbridge, Alberta, Canada
T1K 3M4

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