Re: Tissue processing problems--how was the tissue treated before arriving in the lab.

From:Sharron Ladd

Dear Kathleen,
I work in a small university lab as well. I work by myself, which makes it a little easier to troubleshoot when things go wrong. While it makes perfect sense to question one's protocol and equipment, when tissue comes out rock hard and difficult to section, I generally have to ask, how was this tissue treated BEFORE it arrived in my lab? Usually there is one of 2 problems: improper tissue storage and underfixation. These are things that I usually have no control over and they could both cause the problems you are having.
One thing I do have control over (because I am in a research lab and don't have to have a 24 hour turn around) is the processor schedule. Of course it is easiest to put  the 2x2x1 mm liver pieces in with the 10x7x5mm brain pieces and run them together on the same processing schedule, but this will usually mean that the liver pieces will be dry and difficult to cut and the brain won't be completely infiltrated. If I have the time, I will run the liver by hand, or on a short schedule and I'll program a longer schedule for the brain. Processing takes longer, but in the long run I save time because I don't have difficulty sectioning. I have learned (the hard way) that processor schedules are not "one size fits all."
Three areas to look at:
1. Proper fixation  (many  factors)
2. Proper storage of tissue prior to processing
3. Tailoring the processing schedule to the size and type of tissue
Hope this helps.

Kathleen Roberts (by way of Histonet) 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


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