better kidney bx frozen sections

From:Gayle Callis

Keeping the biopsy moist until SNAP freezing is imperative. 

The cryosat freezes much TOO slowly at -29C, or even on peltier devices at
-40C (whatever they have setup). Snap freezing with liquid nitrogen cooled
isopentane or at Liq N2 temperatures is extremely important to prevent
freezing artifact - this shows up as major separation of kidney tissue
components - this brought major complaints from my pathologist. There is a
lovely device from Shandon (if I remember correctly, and recently discussed
on Histonet) which makes this much easier for clinical laboratories, and
well worth the investment to insure good snap freezing.  

We preferred immersing a freshly cut frozen section into neutral buffered
formalin for 30 seconds or so, then do H&E to examine for glomeruli.  If
more H&E stained FS were needed, these could sit for a day or more without
problems in NBF (when compared to a paraffin section, NBF fixed frozens had
almost identical morphology quality). Frozen sections destined for
immunofluorescent (IFA) staining, are air dried for an hour minimum,
acetone fixed, then air dried overnight before IFA or proceed with IFA
after a shorter 30 minute air dry on same day.  

Whatever you do, do not put a coverslip over a fresh kidney biopsy to
examine for glomeruli, it squashs the tiny round biopsy flat, ruining
morphology, making it a mess for subsequent FS and serial paraffin
sectioning. Use a hanging drop slide with a well in it to examine the
tissue IF you TRY (the key word here) to look for glomeruli.  We found it
far easier to just do FS/H&E to look for glomeruli - mainly because a
technician examining for glomeruli never really saw them grossly and FS was
still the final call!  We had far better luck with the latter.  

Gayle Callis
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)


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