[Histonet] Section thickness in immunofluorecsence studies

From:"George Cole"


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.

georgecole@ev1.net      .            

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