[Histonet] Reply- isopropanol as dehydrator in microwave method

[pooja chopra]

Dear Histonetters,
Thanks for your helpful notes
I am sending a copy of this email on the forum as it will be good histonet 
etiquette.

Dear Diane,
Thanks for  the reply. The size of block is not measured, but I try to be as 
close to a maximum of 1.5X1.0X0.5. Actually with the 5 types of tissues I am 
including in my study, lymph node, bowel, gall bladder, prostatic chips 
(TURP), and ovary(mostly cysts)  mostly my tissue size is much less in size, 
at least the minimum thickness is less than 5 mm in most cases which I 
believe is most important.  Here is my protocol.

FIXATION (stabilization)
immersing the specimen in normal saline and irradiating in the microwave 
oven for 5 to 10 minutes(maximum 15 minutes) I check after 6 minutes and 
then at my discretion. Tissue pieces are kept in microwavable plastic 
cassettes submerged in sufficient amount of saline in 250 ml beaker and 
subjected to irradiation.
PROCESSING:
Isopropyl alcohol (60 %) for 8 minutes (4+4)
Isopropyl alcohol (80%) for 6 minutes (2+4)
Isopropyl alcohol (100%) for 5 minutes(1+4) (generally I open the cassette 
to check at least at this step)
Isopropyl alcohol (100%) for 5 minutes (1+4)
Molten Paraffin wax (bath I) for 5 minutes
Molten paraffin wax (bath II) for 5 minutes

Since my oven has only powers: cook and defrost and all the working is 
possible only on defrost mode, (cook is very high) I have to put water load 
after the initial few minutes of each stage.e.g. (4+4) in 1st step of 
dehydration.

Please review the procedure at your discretion, are you using a domestic 
oven too? Please do let me know the email id of Cheryl Crowder, if possible 
I would like to contact her.

Dear Jeff,
I have tried to lower the power of the oven, by using in defrost mode and 
also by using water load. But I don’t think I can do much about the cycle 
duration, it is fixed in any given model. At times I get very good results 
but consistent good results have only been with prostatic chips. My lymph 
nodes are very unpredictable, once I had actually fixed fresh lymph node and 
processed, all in the microwave with excellent results. But since then all 
my lymph nodes have been jinxed. I would like to know what a PID controller 
is.
Can it be used in domestic oven?

Dear John A. Kiernan,
Can you please let me know what is the Web of Science? Is it a journal or a 
website? I do have the book by Drs Boon and Kok, and am trying to find the 
toolbook by Dr Login, but I have a problem following up all the references I 
find, because some of the specialized journals (especially European) are 
hard to find in the Post Graduate Institute library over here in Chandigarh, 
India.


Thanks a ton,
Pooja

Hi Pooja,

It is actually better to use isopropanol as the final step between the 
alcohol and paraffin, since it has a lower boiling point, it evaporates out 
faster than ethanol, once you have transferred the tissue into the molten 
paraffin.  I don't know what size your tissues are, but why don't you send 
me your protocol with tissue size and if you're having any problems I could 
review it.  In some routine labs, with commercial tissue processors, they 
only use Isopropanol and not ethanol, since they can't afford it.
Best Regards,
Diane
If you are having a problem with your outcomes, it is because of your
microwave and not the use of IPA as a dehydrant or a "clearing agent".
I have used it in a Milestone Lab Oven with great success. Your microwave is
probably too long in duration of the power application at too high a level
(wattage). This was our problem previously too. You need a very short duty
cycle and a low wattage. A PID controller comes in very handy too.
Jeff Jurczak
Dear Pooja:

I have used isopropyl alcohol for many years in processing and staining
without problems.  I've never done microwave processing so I can not comment
on that aspect but I've heard that adequate fixation is absolutely necessary
before doing microwave processing
denise <@t> pclv.net

For almost all applications in practical
histotechnology you can use isopropanol
instead of ethanol. Both these alcohols
mix similarly with water and with clearing
agents such as xylene or chloroform.
Neither ethanol nor isopropanol will mix with
solid or melted paraffin wax.


For a thesis on microwave processing you will
need a copy of Kok & Boon's book as a starting
point, and you will need to follow up their
references. If your library subscribes to
Web of Science you will be able to find recent
papers that cite earlier papers and books.
Without access to Web of Science it is impossible
to find out if someone else has recently done
and published your intended research.

Hope this helps,
--
-------------------------
John A. Kiernan

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