tissue processing -Reply

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From:Carol McCollough <cmccollough@dnr.state.md.us>
To:HistoNet@Pathology.swmed.edu, d.a.faichney@stir.ac.uk
Date:Thu, 11 Mar 1999 10:15:00 -0500

Hello Deborah:

We work with lots of fish, mainly striped bass (Morone saxatilis), white
perch, Atlantic menhaden, Fundulus spp., and catfishes.  

Some of the tissues from bass can be quite large, bisected hearts for
example, and are placed in megacassettes.  We also collect a tissue
block consisting of spine, muscle, skin, and kidney that can be very
large.  We trim everything so that it will fit into the surface area of a
standard cassette, otherwise it won't fit into our embedding molds. 
Smaller fish are serially cross-sectioned from snout to caudal peduncle

All tissues are allowed to fix AT LEAST 48 hours in 10% NBF or alcoholic
zinc formalin (Anatech).  Bony tissues (gills, spine, fin pieces) are then
decalcified in 3 changes of 10% EDTA for 24 hours each and then
washed for 1-2 hours in tap water.

Fixed tissues are placed in 70% ethanol to await processing.

We use an RMC 1530 processor with continuous stirring.  Our 15 hour
overnight schedule is as follows:

70% ethanol           10 min (ususally tissues have been in 70 for several
hours prior to loading the processor, so a longer time in 70 is

80% ethanol             1 hr
95% ethanol             1.5 hr
95% ethanol             1.5 hr
100% ethanol             1.5 hr
100% ethanol             1.5 hr
100% ethanol             1 hr
100% ethanol             1 hr
xylene                         1.5 hr at 35 degrees C
xylene                         1.5 hr at 40 degrees C
Paraplast Plus             1 hr at 59 degrees C under vacuum
Paraplast Plus             1 hr at 59 degrees C under vacuum
Paraplast Plus             1 hr at 59 degrees C under vacuum

I hope you find this useful.

Regards - 
Carol B.  McCollough, HT (ASCP)
Diagnostics & Histology Laboratory Manager
Maryland Department of Natural Resources
Fisheries Service
Cooperative Oxford Laboratory
904 S.  Morris Street
Oxford, MD 21654

>>> Deborah Faichney <d.a.faichney@stir.ac.uk> 03/11/99 07:46am >>>
Hi ,

We work with fish tissues, mainly Salmonids.  Our pathologist has asked
we can reduce the overnight processing to 16 hours from 21 hours,
enabling him to announce urgent samples late afternoon!!

 We use a series of Industrial Methylated spirit, Ethanol, Chloroform then
Paraffin.  Our tissue samples can be quite large (up to 3cmx1cm),
they are below 5mmx5mm.

Any ideas or comments will be gratefully received.

Debbie Faichney
Institute of Aquaculture
Stirling University

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