RE: [Histonet] Section thickness in immunofluorecsence studies

From:"Barry R Rittman"

you have hit upon a common problem. It seems that the tendency has been "the thinner the better". I believe that for histology, there is a need for both thin sections and thick sections. Thinner sections can provide superior reolution but sometimes we lose the wood for the trees. It should be remembered that counting cell numbers in thinner sections is fraught with disaster, the thinner the section the greater the error. Abercrombie discused this a long time ago.
I would urge y'all to repeat George's experience and also try thicker sections and see the difference.
	-----Original Message----- 
	From: on behalf of George Cole 
	Sent: Tue 11/18/2003 12:27 AM 
	Subject: [Histonet] Section thickness in immunofluorecsence studies
	I just visited the hospital I retired from. In talking with the great gal who replaced me and my good friend who runs the immuno lab I mentioned that when I did immunofluorescence studies on kidney, I cut sections thicker than anyone else---I was shaky with  2 or 3 micron sections as a beginner, so I cut one 3 micron, then went up to 5 microns,  and, because I had been doing nothing but muscles and nerves in which I did 8 and 10 micron sections, I included an 8 micron section. Well the kidney turned out to fluoresce with two preps. The 3 micron section was not very dramatic.  The 8 micron section was brilliant, but a bit globby; but to my surprise, all hands chose the 6 micron section.  Also, as I had developed the multiple sections techniques on muscles and nerves, I cut one 3 micron section for slide 1, 2 and 3, then went back and cut a 6 micron section for slide 1, 2 and 3, and did the same for 8 microns sections. Well, I never went back to the 2 or 3 micron sections. I made 6 microns standard----and got many beautiful photos brilliantly shining. The 8 micron section was  a little much, so I used that only with bigger pieces of tissue. But remember what we are doing in this procedure----we are NOT staining fine nuclear detail that takes thin sections to do justice to---we are staining BLOBS and the photos of the  6 microns sections use to be passed around getting OOOHHED and AAAHHHED by the residents. Also, about the multiple sections---this wasn’t always possible, because the kidney biopsies are usually quite small and I had to leave tissue enough to do the Jones silvers----but in this case, of the three sections on one positive prep, only one of the sections lit up. And what did that tell me?  It told me that a single section per slide work up on this case could miss being positive something like 66% of the time.  So I kept doing a 3 micron and as many 6 micron sections cut for each slide as the size of the tissue would allow. The 3 micron section NEVER was positive, by chance or by whatever----and I passed around many brilliantly lighted kidney sections photos of the 6 micron sections.      .            

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