[Histonet] Details of existing protocol

[Barlow, Gillian]

Dear All

Several of you have replied asking for details of my protocol, so I thought
I would post it and ask for comments.  Please note we do not have a
processor, everything is done by hand.  Many thanks for your help, this
seems to  be a really nice group.

Fixation (day 1):  
Fixative is 4% paraformaldehyde, made fresh each time and pH'd to 7.3-7.4
and chilled on ice.  
Hearts are harvested whole, dropped directly into fixative on ice (25-30ml
each) and fixed overnight at 4 degrees.  
Overnight is usually 16 to 20 hours.  I have also tried cutting the hearts
in half before fixing, and substituting 10% phosphate-buffered formalin
(Allegiance) for 4% paraformaldehyde.

Dehydration and embedding (day 2):
10% Ethanol, 2 hours, 4 degrees
20% Ethanol, 2 hours, 4 degrees
30% Ethanol, 2 hours, 4 degrees
50% Ethanol, 1 hour, 4 degrees
70% Ethanol, overnight or until we are ready to process the tissue, 4
degrees

Dehydration and embedding (day 3):
80% Ethanol, 1 hour, 4 degrees
95% Ethanol,1 hour, 4 degrees
100% Ethanol, 1 hour, 4 degrees
Xylene Bath 1, 1 hour, room temp.  
Xylene Bath 2, 1 hour, 65 degrees (usually but not always in vacuum oven)
Xylene Bath 3, 1 hour, 65 degrees (usually but not always in vacuum oven)
Paraffin Bath 1, 1 hour, 65 degrees (usually but not always in vacuum oven).
Paraffin is Paraplast Plus.
Paraffin Bath 2, 1 hour, 65 degrees (usually but not always in vacuum oven)

At this point we usually take the casettes out of the paraffin and allow
them to solidify at room temperature overnight.  The following day, we put
them back in to a third paraffin bath and leave them there for 2 to three
hours (65 degrees with or without vacuum again).  

Then we embed, which for us is the difficult part.  The embedding machine we
have access to is three floors away, so we take the jar of paraffin
containing our cassettes downstairs (transporting in a larger container
containing some hot water to stop the paraffin from solidifying), transfer
the casettes into the holding tank of the embedding machine, give them 10 to
20 minutes to equilibrate, and then embed.

Gillian Barlow, PhD
Postdoctoral Fellow
Laboratory of Julie Korenberg, PhD, MD
Cedars-Sinai Medical Center
Davis Bldg, Lab 2007
110 George Burns Rd
Los Angeles, CA 90048

Phone: (310) 423 7650
Fax: (310) 423 0302
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