RE: How could I get better frozen slides?

From:Tony Henwood

Dear Yichao,

I would transfer the renal biopsies in a cell culture fluid (eg Hanks), place them in an esky with ice if you have to travel some distance.

I recommend you RAPIDLY freeze them in OCT using Liquid Nitrogen or some similar FAST freezing method (I believe the main problem here is the slow freezing offered by the cryostat).

If you are staining them H&E, try immediate methanol fixation of the frozen sections.
If doing immunofluorescence, then air dry for 20-30 minutes prior to staining.
Other comments below.

Hope this helps,

Tony Henwood JP, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital at  Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: (02) 9845 3306
Fax: (02) 9845 3318

-----Original Message-----
From: yichao wu []
Sent: Tuesday, 3 June 2003 12:54
Subject: How could I get better frozen slides?

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? Through
lower temperature is very inconvenient. NO
2) Should we incubate the specimens in sucrose buffer before immerse them in
OCT? How to do it in details? Not necessary
3) Should we change a cyrostat? (Ours is from Bright and have used it for
about 10 yrs) If you rapidly freeze the tissue and the cryostat is working well, there is no need.

Could you kindly give some suggestions? We just wish to have frozen sections
of better quality.Such as could identify basement membrane and glomeruli in
the setions.Now all we could see is a mass of cells. What thickness are you cutting? Try thinner

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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