Re: better kidney bx frozen sections
I agree with all of Gayle's suggestions. This is how I've handled kidney bx
in the past. Freezing quickly in liquid nitrogen or cooled isopentane is the
key. Also, we would put the fresh biopsy under a dissecting microscope to
look for glomeruli. The glomeruli looked like small, round, red puff balls.
This way we knew where to divide up the kidney for EM, routine paraffin, and
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)
> email: firstname.lastname@example.org
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