Re: Tissue processing problems
|From:||Kathleen Roberts (by way of Histonet)|
Sorry, I did post my processing schedule to the list, but for whatever reason
it has not gone through yet, so here it is:
Our standard program is as follows (it's varied according to thickness of
70% EtOH, 2 changes, 25 min in each; 95% EtOH, 3 changes, 25 min each; 100%
EtOH, 3 changes,
25 min each; xylene, 2 changes, 25 min each; paraffin, 2 changes under vacuum,
61 degrees C using
Fisher's Tissue Path Paraplast X-tra paraffin, 45 minutes each.
As for which rodents: mostly rats and mice of various backgrounds. Fish tissue
(zebrafish, to be specific) fixing: also in 10% formalin overnight, then
washed out and placed in 70% EtOH. If we are cutting whole fish, we'll fix
first, wash, then place it in decalcifying solution, wash, then 70% EtOH until
I can put it into the processor.
Thank you for your help!
Principal Lab Technician
Molecular Pathology Facility Core
Dept of Pharmacology & Toxicology
Ernest Mario School of Pharmacy
41 B Gordon Rd
Piscataway, NJ 08854
FAX (732) 445-6905
Gayle Callis wrote:
> You failed to give processing times in your solvents, etc probably
> overdehydrating, overclearing and too long an exposure to paraffin. If you
> add temperature to each processing station - that contributes to
> overdehydration of rodent tissues, not a good thing although manufacturers
> may tell you it works.
> What kind of rodents? How do you fix fish tissues?
> More details please, and fill in with your processing schedule.
> At 09:31 PM 8/12/2003 -0500, you wrote:
> >To all-
> >We are a small university pathology lab that does mostly rodent and some
> >fish tissues, and lately they have been coming out of the processor hard
> >as a rock and very difficult to cut, and we have been trying to figure
> >out what, if anything, we are doing wrong. We have tried changing out
> >the reagents more often, but it doesn't seem to have made a difference.
> >We doubt that it's the temperature of the paraffin, as our settings are
> >in agreement with the manufacturer's recommendations, and the
> >temperature displayed agrees with our thermometer. Could you please send
> >me your processing protocols for comparison?
> >If you like, I will send you our protocol as well. Any and all ideas
> >and suggestions will be very welcome.
> >Thanks in advance for your help-
> >Kathleen Roberts
> >Principal Lab Technician
> >Neurotoxicology Laboratories
> >Molecular Pathology Facility Core
> >Dept of Pharmacology & Toxicology
> >Ernest Mario School of Pharmacy
> >Rutgers University
> >41 B Gordon Rd
> >Piscataway, NJ 08854
> >(732) 445-6914
> >FAX (732) 445-6905
> 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: email@example.com
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