Thank you for your help as to " How could I get better frozen slides?"

From:yichao wu

Dear Tony,

I forget to mentioned that we cut 4um frozen sections.And as liquid nitrogen 
is not very convenient for the technician in our routine work.I just wonder 
if we could put the OCT-specimen in a -70C fridge for quick freezing?

And could I do as below,
Fix the fresh specimen in a kind of fixation solution first(like ethanol) 
and then transfer it to the lab at 4C.
Immerse the specimen in OCT on a metal holder.
Put them all in -70C immediately.
After OCT is frozen,transfer them quickly into the cyrostat which adjust to 
-18C and then sectioning.

Any comments are welcome.

Thank you very much!

Yichao WU
Jinling hospital
Nanjing China

>From: Tony Henwood 
>To: 'yichao wu' ,
>Subject: RE: How could I get better frozen slides?
>Date: Tue, 03 Jun 2003 14:45:11 +1000
>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
>If doing immunofluorescence, then air dry for 20-30 minutes prior to
>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 
>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 
>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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