Fwd: RE: Re: [Histonet] processing fatty tissue

From:Rene J Buesa

For "general enjoyment" from 2 "old timers".
  James McCormick and RenÚ J.

Rene J Buesa  wrote:
  Date: Fri, 1 Jun 2007 07:18:42 -0700 (PDT)
From: Rene J Buesa 
Subject: RE: Re: [Histonet] processing fatty tissue
To: "McCormick, James" 
CC: Doug Martin , 
"Drew N. Mehta" , 
Lamar Jones , Maxim Peshkov 

  Dear Dr. McCormick:
  I could not have said it better! Your description of tissue components being GENTLY awashed by succesive flows of chemicals in low gradients of different chemical actions is what exactly exists in any procedure that intercalates between the different steps leading to the substitution of tissue water with the infiltrating medium.
  That is the rationale behind using EthOL at increasing concentrations from a "quite shocking" 60% start (could be gentler, although TAT imposes restrictions and limitations on "tissue processing gentleness").
  Once the tissues have been "completely traumatized" at the end of the dehydration, the following "chemical shock" is given by the antemedium, usually the "infamous and noxious xylene" to end with the "soothing" effect of the paraffin.
  That is the rationale of my procedure of eliminating the harsh xylene effect with the combination of alcohols and "liquid paraffin" = mineral oil. In my procedure by mixing ethanol + isopropanol + mineral oil, and in Maxim's modification by mixing propanol and mineral oil only.
  Nothing new about using propanol, it has been in use to dehydrate since the early twentieth century, and now recently has been "resurrected" as the dehydrant of choice and sole chemical between the tissue water and the wax, as in the technology in use by the Peloris
  instrument which uses hight "instantaneous" temperatures to "dry-out" the tissue to eliminate the propanol and leave it "open" to the wax influx.
  I personally would never treat a tissue sample so harshly but you know how tissue preferences go, they are like beauty, all in the beholder's eyes.
  For me tissue processing should be gentle though.
  Being an "old timer" like me, you should remember the fantastic results we used to obtain when clearing tissue with Canada balsam, the precaution to cover the floating dehydrated tissue with a piece of filter paper moisted in ethanol to prevent the tissue to dry-out, and you should also remember that the tissue itself "said" when "I am ready for infiltration", after going to the bottom of the container, before being quickly washed with benzene. Remember those days? And also how long it took to infiltrate? And how soft they were to section?
  Again TAT chastised all of us and dictated quicker but NOT better procedures.
  Mixing alcohols and mineral oil, during protocols that take the same time as with other methods that include xylene, ease infiltration and help the histotech to section better, thinner and get rid of xylene as well.
  Just a thought you "provoqued" with your dancing image!
RenÚ J.

"McCormick, James"  wrote:
  Rene J.
As I am familiar with much of the "antique" instruments and tissue 
processing methods............your writing helps me to understand the 1860's use of essential oils to dehydrate and clear tissue for "tallow" infiltration and later paraffin waxes.....one molecule after another "holds hands and changes partners" that we might call "the line dancing of tissue processing for histotechnology" .
Kindest regards, 

-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Rene J Buesa
Sent: Friday, June 01, 2007 7:53 AM
To: Maxim Peshkov; histonet@lists.utsouthwestern.edu
Subject: Fwd: Re: [Histonet] processing fatty tissue

My answer to Robert without the attachment.
RenÚ J.

Rene J Buesa wrote: 
Date: Fri, 1 Jun 2007 05:48:45 -0700 (PDT)
From: Rene J Buesa 
Subject: Re: [Histonet] processing fatty tissue
To: Robert Chiovetti 

The procedure is attached. The fundament is a GENTLE dehydrant substitution by the infiltrating agent (paraffin) and ELIMINATES the use of an antemedium (xylene).
The general function of an antemedium is the ability to be the "connection" between the dehydrant and the wax (paraffin) because it mixes with both.
Xylene (as well as white naphtha or some other aromatic chemicals) can do this, BUT the thing is that mineral oil (MO) IS paraffin of a low molecular weight so the antemedium is not needed or a mixture of MO with alcohols constitutes the antemedium.
My procedure uses EthOL to dehydrate and later the antemedium is substituted by the mixture of EthOL + Isopropanol + MO
Maxim has simplified the procedure because he process manually and don't have the advantage of vacuum or pressure or agitation as I did when developed the method.
So it turns out that Maxim's modification is easier and more direct; he just dehydrates with propanol and later goes into the gentle substitution with a mixture of 5 parts of propanol + 1 part of MO heated at 50║C followed by another mixture of 2 parts of propanol + 1 of MO heated also at ║C to obtain the gentle and complete infiltration of ANY type of tissue.
The infiltration with MO gives the tissues a softness never achieved with any other antemedium. You will see when you try it.
Maxim's method is simpler than mine and, in the long run, will be more acceptable to all histotechs and also meets the objective of eliminating xylene from the histology lab.
The procedure uses 2 chemicals that are cheaper and when needing to be disposed off, the propanol can be evaporated, the used MO mixed with used paraffin and both disposed off as a solid, cutting costs also in disposal.
Try it, you will like it! 
RenÚ J.

Robert Chiovetti wrote: 
Maxim, Rene (and Other Histonetters),

That's interesting re: using either a mix of ethanol+isopropanol+mineral oil (Rene) or isopropanol+mineral oil (Maxim) for breast tissue. Could you share your recipes with us? 

I have a customer (derm path) who could probably benefit from this for larger and thicker skin specimens which sometimes have a lot of subcutaneous fat associated with them.

Thanks in advance, if you could share the recipes!



Robert (Bob) Chiovetti, Ph.D.
Southwest Precision Instruments
The Desert Southwest's Microscopy Resource
Tucson, Arizona USA
Tel./Fax 520-546-4986
Member, Arizona Small Business Association

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