About "better frozen sections"

Dear Dr.Calllis, Thank you very much for your kind help all the time.Here is my answer for your question about

>I have a question for you.Are you trying to get good morphology and still do LCM on the same tissue section? If so, you will NOT have the best of both worlds...

Here the biopsy specimens are tiny.If we want to have any glomeruli in every frozen sections,we have to try to combine immunohistochemisty and laser microdissetion together.

I should explain further, the biopies here are aparted for four purposes: formalin-fixed paraffin-embeded sections for light microscopy; acetone/methanol-fixed paraffin-embeded sections for immunohistochemistry (since we found that this kind of fixation maintain antigens well and have very good morphology as well. And we transfered from frozen section to this kind of section to do IHC in 1999); frozen sections and lastly, another part for electron microscope.So the morphology analysis by light microscopy is apart from immunohistochemisty.

Most time it is hard to guarantee there is any glomeruli in the frozen section part.So our director wants that the second part and the frozen section part fusion into one part. That is to say, she wants again to do IHC on frozen sections.But she don't want to give up the good morphology of such paraffin-embeded sections.However she also knows that such paraffin sections are not suitable to do some molecular analysis such as laser microdissection.Hence she is very very hesitate!

So now I am responsible for better frozen sections,especially better morphology.I realized that there may be something wrong with our frozen section protocol.So I asked and get much help from histonet.Now I add "quick-freezing" by liquid nitrogen as you know,and have better results.But it still could not be comparable to paraffin sections.

Therefore,on one hand,we are trying to do laser microdissection on such acetone/methanol-fixed paraffin-embeded sections,wishing to acquire RNA(But we tried and tried and failed). On the other hand, I still want to improve the frozen section protocol further for better morphology to the uppest level.I wish someday my director would like to transfer to frozen sections for both IHC and molecular analysis.Maybe the morphology would be a little worse, but we have better material for laser microdissection and in situ hybridization.

I wish my explanation is clear enough.Sorry for the possible misleading. And I wish you could give me more instructions on my problems.We are really in dilemma!

Thank you very much again!

Yichao WU

Jinling Hospital

Nanjing China

>From: Gayle Callis

>To: "yichao wu"

>Subject: Re: I Got acceptable frozen sections with L N2 and wait for your comments

>Date: Tue, 10 Jun 2003 08:52:56 -0600

>A cryomold is what you embed tissue in - to form a block. These come in

>metal or disposable plastic - the plastic molds are best for snap freezing.

>The chuck is what holds the block when cutting in cryostat. The block is

>mounted on this chuck, then placed into the microtome holder prior to

>sectioning. DO NOT USE THE CHUCK FOR SNAP FREEZING, it is

>counterproductive for LCM purposes. The instructions I sent yesterday were

>from two ladies who do LCM all the time. They DO NOT USE CHUCKS FOR SNAP

>FREEZING.

>If you do prefixation and cryoprotection, you will ruin the RNA! DO NOT DO

>THAT! Hopefully, your sodium chloride solution was made up with RNAse free

>water.

>You should cut your biopsy at -20C. Quit trying to mess around with

>sectioning, and just set cryostat to minus 20C, equilibrate the block to

>same temperature and cut. -29C is too cold, and -18C is only 2 degrees

>from -20C. I cut hundreds of kidney biopsies at -20C and never had problems.

>You needed to tell people on Histonet what you wanted to do, in other

>words, Laser capture microdissection. This is a very fussy protocol, and

>messing around with PFA and sucrose will not get you the results you want,

>RNA will be eaten by RNAse that is found everywhere.

>I have a question for you

>Are you trying to get good morphology and still do LCM on the same tissue

>section? If so, you will NOT have the best of both worlds. For light

>microscopy, the section needs to have a coverslip mounted, for LCM the

>section must be as dry as possible for LCM, meaning DO PUT A COVERSLIP ON

>THE SECTION.

>To get perfect morphology on a frozen section for light microscopy only,

>cut a frozen section and immerse it immediately into neutral buffered

>formalin and go back later to do the H&E staining. If you want LCM, you

>need to cut the section, put it in a box ON DRY ICE (you must preserve the

>RNA or have no results). These sections that are put on dry ice must be

>fixed in 70% ethanol for 10 seconds for LCM. 70% ethanol is a poor

>fixative if you want to do immunostaining in conjunction with LCM. You can

>use pure acetone. In order to correlate light microscopy and LCM, you can

>do adjacent serial sections, one stained for light microscopy and adjacent

>section for LCM. If you do immunostaining before LCM, then you cannot put

>a coverslip on top of that section!

>What ever you do, quit insisting on PFA fixation and sucrose, it will get

>you poor LCM/RNA ISH results!

>Frozen sections will never have perfect morphology, mainly because they ARE

>frozen sections and not processed into paraffin after formalin fixation.

>The main reason you are getting bad morphology is the slow freezing that

>still exists, and that huge metal chuck is still a problem. It takes too

>long to cool down a large piece of metal. Learn to snap freeze the biopsy

>embedded in OCT with Liquid nitrogen. It only takes 10 seconds or less,

>not 1 minute! I would never use a chuck to freeze a biopsy, it is to much

>metal that needs to be cooled, it is better to freeze the biopsy in OCT,

>then MOUNT THE BLOCK ON THE METAL CHUCK before sectioning. We do all

>frozen section RNA ISH that way here without problems.

>The people on Histonet assumed you were doing these biopsies for diagnostic

>purposes for renal disease, and not LCM. In the future, provide details.

>Most people do NOT do LCM which requires special handling of the biopsy. I

>did not know about your LCM until after several replies. Consequently,

>Tony Henwood probably assumed you were working with a diagnostic situation

>for renal disease.

>Gayle Callis

>MT,HT,HTL(ASCP)

>Research Histopathology Supervisor

>Veterinary Molecular Biology - Marsh Lab

>Montana State University - Bozeman

>S. 19th and Lincoln St

>Bozeman MT 59717-3610

>406 994-6367 (lab with voice mail)

>406 994-4303 (FAX)

>email: gcallis@montana.edu