frozen sections

From:yichao wu

I was told that sucrose could dehydrate the tissue and protect from ice cystals forming. Yes I will try to avoid it next time.

Up to now,I get the impression that snap freezing is the most important, right? So, may I do as follows(Could I use -70C fridge instead of solid ice)?

1. Put fresh kidney biopsy specimen in a Hank's BBS at 4C and transfer to the lab.

2. Precool isopentane in -70C fridge in small vials for several hours. Immediately put specimen into isopentane for 30 seconds.

3. Precool chuck in -70C fridge.Put a drop of OCT on the top of chuck and immediately transfer the frozened specimen to OCT and let them all frozen for 1 minutes.

4. Transfer them to -18C cyrostat and wait for 5 minutes.

5. Start to sectioning.

Could you kindly make some comments on this procedure?

 

Yichao WU

Jinling Hospital



 

>From: Tony Henwood
>To: "'yichao wu'" , Tony Henwood
>CC: histonet@pathology.swmed.edu
>Subject: RE: Thank you and still the shrink problem
>Date: Thu, 5 Jun 2003 14:31:18 +1000
>
>Why do you need to infiltrate with sucrose? You probably do not need to
>
>Tony
>
>-----Original Message-----
>From: yichao wu [mailto:yichaowu@hotmail.com]
>Sent: Thursday, 5 June 2003 13:40
>To: AnthonyH@chw.edu.au
>Cc: histonet@pathology.swmed.edu
>Subject: Thank you and still the shrink problem
>
>
>
>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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> > > >
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> >Children's Hospital at Westmead
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>intended solely for the use of the individual or entity to whom they
>are addressed. If you are not the intended recipient, please
>delete it and notify the sender.
>
>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
>
>This footnote also confirms that this email message has been
>virus scanned and although no computer viruses were detected,
>the Childrens Hospital at Westmead accepts no liability for any
>consequential damage resulting from email containing computer
>viruses.
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