Re: [Histonet] Silly question about deparaffinization

From:Paul Bradbury

Hi Eva,

Assuming that you are dealing with routine 4 micron sections of paraffin 
wax, a room temperature of 18-21 C  (65-70 F for you guys South of the 
49th), reagents that are changed on a regular basis, adequate volumes of 
each reagent, and some form of agitation, you can afford to make some 
major cuts in your timing.

Assuming that all the above criteria are in place, I would suggest the 
Xylene    5 minutes
Xylene    5 minutes
Absolute alcohol    2 minutes
Absolute alcohol    2 minutes
95% alcohol     2 minutes
70% alcohol can be skipped without any harm to the tissues.
Water    1 minute
Carry on with staining procedure of choice.

For routine staining, the above times will work just fine. While it is 
essential that all traces of wax are removed, 30 minutes in xylene is 
grossly excessive, Once the xylene has removed the wax, the time spent 
in alcohol does not need to be very long. All you are trying to do is 
replace one fluid with another ... and that happens pretty quickly ... 2 
minutes in each alcohol is ample. The most contaminated reagents should 
be replaced each day or on a regular basis, or if the reagents begin to 
look contaminated, discard them.

There are references in the literature to methods that require prolonged 
treatment with solvents prior to staining. The original procedure for 
the Lendrum's MSB fibrin stain specified 24 hours treatment with 
trichlorethylene to "degrease" the sections, but I don't know anybody 
who still does this!

Paul Bradbury
Kamloops, BC

PS ... What are you going to do with all this spare time ?

Eva C Andersson wrote:

> Good morning,
> I am probably asking a very silly question but I recently spoke to 
> someone about deparaffinization and hydration. I found that the times 
> that they use and the times that we use are very different. So my 
> question to you is this: What lenghts of time do you use for Xylenes, 
> 100% alcohol, 95% alcohol and 70% alcohol?
> It is about deparaffinising charged (+) slides. So nothing fancy.
> Just curious. Maybe I can cut back some on the timed that we use which 
> are:
> 30min Xylenes, 10min 100%, 10min 95% and 6min 70%.
> Thanks for your responses.
> Eva Andersson
> Georgetown University
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