From: | Pamela Marcum |
-----Original Message-----
From: yichao wu [mailto:yichaowu@hotmail.com]
Sent: Sunday, June 08, 2003 2:54 AM
To: pmarcum@polysciences.com
Cc: histonet@pathology.swmed.edu
Subject: Dr.Marcum Sucrose combined with the fixative?
Thank you very much! I am really a layman.And your message is very useful to me!
As you mentioned," The 30% sucrose in the fixative is to stabilize the tissue and prevent the holes you are currently getting".I just wonder should I use sucrose combined with the fixative?
You know I just incubated the specimen firstly in PFA and then in sucrose.Do you mean I should put it in a mixture of 30% sucrose and the fixative?
Thank you. Yichao WU
>From: "Pamela Marcum"
>To: "yichao wu" >Subject: RE: Thank you and still the shrink problem >Date: Thu, 5 Jun 2003 08:52:03 -0400 > >the point to -70C is to very quickly freeze the specimen. The -20C will be >too slow and may allow more of the same problem you are seeing now. The 30% >sucrose in the fixative is to stabilize the tissue and prevent the holes you >are currently getting. This is not a dehydration step. It is an >infiltration step. I believe others will give you better times however the >whole piece of tissue is in sucrose not the sections. Ten minutes is not >long enough for the tissue specimen to fix and infiltrate. Pam Marcum > -----Original Message----- > From: yichao wu [mailto:yichaowu@hotmail.com] > Sent: Wednesday, June 04, 2003 11:40 PM > 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 > > > > > > > >_________________________________________________________________ > > > >Add photos to your e-mail with MSN 8. 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