[Histonet] Hand processing mouse tissues

[Gayle Callis]

First of all you should invest in three things, a vacuum oven that heats, 
and a Wheaton O ring vacuum dessictor and a  vacuum pump.  You can do mouse 
tissues in one day with the following schedule.. My thoughts are you are 
overdehydrating and exposing your mouse tissues to too much heat.

Fixation is complete.  Use the vacuum dessicator, Wheaton O ring is 
perfect, no grease is needed.  Do all steps under vacuum at 20 psi.

Flex 70         30 min
Flex 95 30 min
Flex 95 30 min
Flex 100        30 min
Flex 100        30 min
Clearite        30 to 45 min RT
Clearite 3      30 to 45 min RT
You do not need a Clearite/paraffin mixture!  And do not add heat to 
Clearite steps!! You are probably cooking your tissues with too much heat, 
particularly overnight in paraffin.
Paraffin        30 min  no more than 60C.
Paraffin        30 min
Paraffin        30 min
Paraffin        30 min (no more than two hours total in paraffin)

This is a 5 1/2 hour schedule, within a working day.  The inexpensive items 
for having vacuum in all steps will speed up your processing of lean, tiny 
mouse tissues.  You did not say, but I am reading between the lines your 
tissues are very dry and friable.

If your tissues seem a bit under-dehydrated, increase time in one 95% and 
in one 100% to 45 min.  Be aware of what tissues you are processing, you 
may want to change the schedule a bit for denser, or larger pieces, liver, 
whole brains, etc.   Tiny nodes will take possibly even less time.

Use a vacuum oven for all your paraffin infiltrations.  Just have beakers 
of melted paraffin, drop cassettes into this, hopefully you have a 
wire/mesh basket to speed up transfer of cassettes, an old Technicon basket 
will work nicely.  or tie them into some cheesecloth for carrying from one 
container to another.

At 11:20 AM 9/29/2004, you wrote:
>It's mouse tissue.  Here's the way I've been doing it recently. I've made 
>changes from my original protocol.
>
>Flex 70 (made from Flex 80).........1 hour (2 times)
>Flex 95.......................................45 min
>Flex 95.......................................1 hr
>Flex 100.....................................1 hr (2 times)
>ClearRite 3..................................1 hr @ Room Temp
>ClearRite 3..................................1 hr @ 60 degrees
>ClearRite 3/Parafin.......................1 hr @ 60 degrees
>Parafin........................................Overnight (It's my stopping 
>point)
>Parafin........................................1 hr
>Parafin........................................1 hr
>
>Please let me know if I need to change anything.
>
>
>Jennifer K. Sipes, RALAT
>Sr. Laboratory Technician
>Johns Hopkins University
>Ross 929
>720 Rutland Avenue
>Baltimore, MD  21205
>phone:  410-614-0131
>cell:     443-413-0853
>e-mail:  jengirl1014 <@t> yahoo.com
>
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)