Could I put the chuck into liquid N2 directly with specimen embeded?

Thank you very much.

And actually I put the PFA solution in a 4C box and incubate the fresh specimen immediately in the solution when it was taken from patient.

And last Friday I tried on kidney specimens from rat.This time I use 0.1M PBS to make 4% PFA fixative.And still had bad results.The fixation and 20-30%sucrose incubation seem to cause the tubules to be apart from each other.(I tried 2 hrs fixation and 30 mins sucrose incubation) There were many holes or spaces in the tissue.Or to say,the tissue is not consecutive.As a control,fresh specimen immediately sectioned in the cryostat had better result.The tubules are connective.

Another specialist replied to me and said, sucrose is used as a mixture in fixative.Really?

I want to try a method next time.I would take back fresh specimen with Hank's solution.And immediately embed it in OCT on the top of a chuck.Then put the chuck into liquid N2 directly with the help of a long forceps.After quick freeze for several minutes,I would get out the chuck and put it into the -40C cryostat and wait for a while.Then sectioning begins.Do you think it correct?

Yichao

>From: frater@ruca.ua.ac.be

>To: "yichao wu"

>Subject: Re: About quick freezing and shrink

>Date: Thu, 5 Jun 2003 15:37:38 -0700

>Dear Yichao,

>I read your procedure and your results and I tried to figure out why

>your morphology still is unsatisfactory. I can give you some additional

>comments in the hope they can help to improve your results.

>First I'd like to mention that I never use NaCl in fixatives. I use 4 %

>Paraformaldehyde in phosphatebuffer 0.05M - 0.10 M pH 7.4.

>I don't think that changing the PFA concentration from 4 % to 2 % will

>make a difference. I don't have experience with tiny kidney biopties,

>but 10 minutes fixation is rather short. Maybe you could try 30 min or

>so.

>Fixation in PFA should give you better results than Aceton/Methanol

>because normally shrinkage is much higher in Aceton and Methanol

>than in PFA.

>Also the 10 min in Sucrose is short since the sucrose solution should

>prevent any freezing artefacts. Maybe you could also try 30 min or

>so. The concentration of sucrose 20 or 30 % normally makes not a

>big difference.

>About the freezing procedure itself I can say that I used different

>methods. If the tissue is well protected by the sucrose, it doesn't

>make much difference what kind of method you use. At the moment I

>freeze my tissue in the cryostat at -40 #176#C. Before I used dry ice and

>isopentane/liquid nitrogen.

>I'd like to ask you if there is a time gap between the moment of taking

>the biopsy and the moment of starting the fixation?

>With kind regards,

>Frank