RE: merosin and emerin on frozen muscle sections.

From:

"Houston, Ronnie"

Manal,

Your freezing protocol will give you disastrous results for any muscle work. Your tissue will be full of ice-crystal artifact. The only way to freeze skeletal muscle is to snap-freeze in -160C precooled isopentane in liquid nitrogen.

Having said that, merosin and emerin immunohistochemistry technique will not as such be affected, just your morphology. You may get some "rings" aroung the ice crystal holes inside the fibers. Emerin is a nuclear marker.

Without more details about your procedures and source of reagents I can't offer any other suggestions.

Ronnie Houston

Regional Histology Operations Manager

Bon Secours HealthPartners Laboratories

5801 Bremo Road

Richmond, VA 23226

(804) 287 7972

-----Original Message-----
From: manal galal [mailto:galalmkh@yahoo.com]
Sent: Wednesday, June 04, 2003 9:45 AM
To: histonet@pathology.swmed.edu
Subject: merosin and emerin on frozen muscle sections.

Dear colleagues,

             My work is on frozen muscle. I am having trouble with merosin and emerin immunostaining on frozen muscle. I wonder if my freezing process is to blame?

   My freezing procedure is as follows:

1-We recieve the specimen immediately after the biopsy is taken.

2-We put it in a fridge for 15 min to relax.

3- The section is stuck on a pre-frozen chuck with a dab of OCT.

4- It is then immediately placed on the quick freeze shelf (-40C) of the cryostat for 15 min.

5-Then the specimen is put in the main compartment of the cryostat        (-20C) and left there for 20 min. to accomodate to the temperature.

6- The sections are then cut at 8 microns and placed on the slides.

7- The slides are left to dry overnight at room temperature.

By the way we get very good results with Dystrophins and Sarcoglycans, using this method of freezing.

Any suggestions??

Thank you

Manal Galal



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