Re: [Histonet] Details of existing protocol

From:Geoff McAuliffe

Dear Gillian:

    Your tissue is over-processed. Very, very over-processed.

1. Fix at room temperature, not cold. For most things cold fixation is a 
myth, it just slows down the reaction of the fix with the tissue.
2. Cut the heart in half and fix in a volume of fix at least 10X that of 
the tissue. If you are not going to cut the whole heart, why fix the 
whole heart? If you are going to cut the whole heart your results will 
be better if it is cut in half for processing.
3. Fix longer, 48 hours is the minimum for full fixation with 
formaldehyde. Depending on what you are looking for there may be better 
4. After fixation wash the tissue in running water for an hour or more.
5. Go directly to 70% ethanol. Any alcohol lower than 50% is a waste of 
time and money.
6. Dehydrate in ethanols at room temperature: 95% 1 or 2 changes, 1 hour 
each, 100% 2 changes one hour each, a 1:1 mix of 100%:xylene one hour, 2 
changes of xylene. No hot xylene!! Also consider using cholorform as a 
clearing agent, it is easier to remove with paraffin.
7. Melted paraffin, 3 changes 30 minutes to one hour each. The was 
should be just a few degrees above its melting point, 65 degrees is 
probably too hot. A vacuum oven is good.
8. You don't need a "machine" to embed. Get some "peel-a-way" molds, 
pour the wax in, wait a 15 seconds, put the tissue in, put the mold in 
cold water in a dish, put the dish in the refrigerator. Make your own 
"molds". Get a block of wood 3X bigger than the tissue. Fold some 
notebook paper around the block to make a box with the top open. Pour in 
hot wax. Wait 15-20 seconds for the bottom to set up a bit. Put tissue 
in. In a minute or two, put block in cold water as above.
9. Make sure your knife is sharp.

Good luck!

Barlow, Gillian wrote:

>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
>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

Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

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