Thank you and still the shrink problem

From:yichao wu

Thank you very much,Tony!

Then I understand the need of "quick freezing" to -70C.I wonder could I only quick freezing to -20C by isopentane? I mean put isopentane at -20C and frozen specimen by it.

Since I once realized that ice crystals form mostly between -30C to -40C.I wonder if I section immediately,not for preservation the specimen,could I only quick freeze to -20C by isopentane?

The other question is, when I use 30% sucrose to dehydrate the specimen for 10 mins, there are much space between the tubules in the specimen.All the tubules apart from each other. But the tubules themselves maintain very well.The cells are clearly seen. How could I avoid this to happen? The "shrink" phenomenon.

Thank you for your kindness!

Yichao

 

 

 

 

 

>From: Tony Henwood
>To: "'yichao wu'"
>Subject: RE: Is it necessary to freeze to(C70C if I would like to section immediately
>Date: Thu, 5 Jun 2003 08:41:10 +1000
>
>Yichao,
>
>From years of experience, stopping the formation of large ice crystals (that
>will occur if you slow freeze) will disfigure your tissue. Since the tissue
>would have been frozen quickly to -70C then you will only have VERY small
>ice crystals. Warming up to -20 or -29C (which seems too cold for cryotomy)
>will not affect the tissue. Water will still remain frozen as small crystals
>at -20C (and even -1C)
>
>Tony
>
>-----Original Message-----
>From: yichao wu [mailto:yichaowu@hotmail.com]
>Sent: Wednesday, 4 June 2003 20:54
>To: AnthonyH@chw.edu.au
>Subject: Is it necessary to freeze to(C70C if I would like to section
>immediately
>
>
>
>Dear Tony,
>
>I don#161#Ot quick freeze the specimen with (r)C70C isopentane or dry ice today.
>The reason is, I could not get access to them today.And the other reason is
>that,since I want to section immediately(just after I obtain the specimen).I
>don#161#Ot know why I should freeze the specimen to (r)C70C or below, then thaw it
>to (r)C29C to section. Wouldn#161#Ot the freeze-thaw be destroyable? (Is it
>necessary to freeze to (r)C70C if I would like to section immediately, not for
>preserve )?
>
>My procedure today was,
>1. put fresh biopsy material in 4% PFA/PBS for 10 minutes (at 4C)
>2. rinse with PBS twice for 10 minutes (at 4C)
>3. place the specimen in 30% sucrose/0.9%NaCL for about 10 minutes when
>the specimen sink to the bottom.(at 4C)
>4. place a drop of OCT on a cold metal holder, then place the specimen
>at the top of OCT.
>5. place the metal holder together with OCT-specimen in a (r)C20C fridge
>waiting for the OCT to be whiten and frozen.
>6. transfer the them all to the cyrostat at (r)C29C and sectioning.
>
>My result is : I could see glomeruli well. But all the tubules #161##176#shrink#161##177#
>very much. There are some space between every tubules.
>I guess the PFA as a fixation and the 30% sucrose as a dehydration all could
>cause the specimen to shrink. Right?
>
>
>Would you kindly give me some suggestions more?
>
>Yichao
>
>
>
>
> >From: Tony Henwood
> >To: 'yichao wu' , Tony Henwood
> >CC: histonet@pathology.swmed.edu
> >Subject: RE: Thank you for your help as to " How could I get better frozen
>slides?"
> >Date: Wed, 04 Jun 2003 09:53:14 +1000
> >
> >It won't freeze very quickly.
> >Maybe you could try useing as little OCT as you can and prefreeze your
>chuck
> >before placing the OCT and specimen onto it.
> >
> >Freezing should take a maximum of 3 seconds.
> >
> >Tiny
> >
> >-----Original Message-----
> >From: yichao wu [mailto:yichaowu@hotmail.com]
> >Sent: Tuesday, 3 June 2003 23:27
> >To: AnthonyH@chw.edu.au
> >Cc: histonet@pathology.swmed.edu
> >Subject: Thank you for your help as to " How could I get better frozen
> >slides?"
> >
> >
> >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' , histonet@pathology.swmed.edu
> > >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
> > >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
> > >
> > >http://www.histosearch.com/homepages/TonyHenwood/default.html
> > >
> > >http://us.geocities.com/tonyhenwoodau/index.html
> > >
> > >
> > >-----Original Message-----
> > >From: yichao wu [ mailto:yichaowu@hotmail.com
> > >]
> > >Sent: Tuesday, 3 June 2003 12:54
> > >To: histonet@pathology.swmed.edu
> > >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.
> > >Sectioning.
> > >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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>Views expressed in this message and any attachments are those
>of the individual sender, and are not necessarily the views of The
>Children's Hospital at Westmead
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>virus scanned and although no computer viruses were detected,
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