Re: How could I get better frozen slides?

From:yichao wu

Thank you very much.

I just wonder if 4% PFA could be used as fixative solution? And how do you prepared isopentane at -70C? I means what is the container of isopentane? Is beaker OK?

And you mean frozen the specimens firstly in isopentane at -70C then immerse it in OCT? I just wonder how do you place the specimens in isopentane? By forceps? You know the specimens are very small and a beaker of isopentane is huge to it.Could you kindly explain it in details?

Thank you very much! Sorry for the interruption and I am really a new comer to frozen sections.




>From: "Lisa Black"
>Subject: Re: How could I get better frozen slides?
>Date: Tue, 03 Jun 2003 10:25:11 -0400
>The kidney biopsies for immunofluorescence are placed in Zeus fixative
>(Michel's fixative) by the surgeon. It is kept at room temperature
>until arriving in Surgical Pathology. The specimen is rinsed in PBS for
>30 minutes and then is placed in isopentane at -70 C. Once frozen the
>specimen is quickly mounted onto a chuck with a minimal amount of OCT.
>This reduces freeze artifact.
>Lisa Black
>Carilion Consolidated Laboratory
>Histology Supervisor
> >>> yichao wu 6/2/03 10:54:20 PM >>>
>In our lab,frozen sections from kidney specimens always look poor.I
>want to
>know how other labs do.
>Our procedures are as below,
>Firstly we get specimens from clinic bedside.
>Take them back to our lab in a 4C box.
>Immerse them in OCT.
>Put directly in -29C cyrostat and wait for them to be frozen.
>Use slides in room temperature to stick to those sectioned tissue. And
>frozen sections are prepared.
>I wonder,
>1) Should we use lower temperature when transfering the specimens?
>lower temperature is very inconvenient.
>2) Should we incubate the specimens in sucrose buffer before immerse
>them in
>OCT? How to do it in details?
>3) Should we change a cyrostat? (Ours is from Bright and have used it
>about 10 yrs)
>Could you kindly give some suggestions? We just wish to have frozen
>of better quality.Such as could identify basement membrane and
>glomeruli in
>the setions.Now all we could see is a mass of cells.
>Thank you very much!
>Yichao WU,Ph.D candidate
>Research Insititute of Nephrology,Jinling Hospital
>Medical School,Nanjing University
>Nanjing 210002
>PR China
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