Re: [Histonet] lipid preservation in paraffin and mineralization of tissues?

From:John Kiernan



Thank you Nicole, for bringing everyone's attention to these two publications. both describe the same technique of introducing highly unsaturated lipids into fatty materials already present in the tissue, and then rendering the unsaturated compounds insoluble in organic solvents by treatment with 2% aqueous chromium trioxide ("chromic acid") solution. The authors do not say why they used chromic acid for this purpose, and their only references are brief and incomplete quotations from books by Pearse and Lillie. 

The usual reagent for chromation of unsaturated lipids is not 2% chromic acid, which probably would remove pathological calcifications and RNA because its pH is 1.1. In lipid histochemistry, chromation is done with potassium dichromate, in solutions of pH 3.5 and higher. Baker's chromation method for formaldehyde-fixed blocks of tissue (Quart. J. Microsc. Sci. 87:441-470, 1946) consists of immersion in 5% potassium dichromate with 1% calcium chloride for 18h at room temperature, followed by 24h at 56-60C and then an overnight wash in running tap water. (The Ca ions were there to improve the precipitation of phospholipids.) In Elftman's "controlled chromation" (J Histochem. Cytochem. 2:1-8, 1954) the solution is 2.5% pot. dichromate at pH3.5 for 18h at 56C - conditions found to be optimal for insolubilizing phospholipids. The lipids may be stained either with a solvent dye such as Sudan black B or by virtue of the bound chromium(III) formed by reduction of dichromate. These are excellent methods for staining myelin and other phospholipid-rich structures such as mitochondria.

The ingenious feature of the Tracey & Walia method is the initial infiltration with en emulsion of linoleic acid and phosphatidylcholine, which allows the localization of the site of any type of lipid by staining paraffin sections with a solvent dye (in their case, oil red O). One would expect the Tracey & Walia method to work even better with a rational chromation procedure than with chromic acid, and with less damage to sites of calcification. I do not know if enough mRNA would survive to allow in situ hybridization to sections of chromated tissue. If no-one else knows, you will just have to try it.

John Kiernan
Dept of Anatomy & Cell Biology
University of Western Ontario
London, Canada
= = =
----- Original Message -----
From: Nicole Collette 
Date: Monday, November 10, 2008 12:23
Subject: [Histonet] lipid preservation in paraffin and mineralization of tissues?
To: histonet@lists.utsouthwestern.edu

> Hello, All,
> 
> I am a postdoc with a couple of self-taught years 
> of histology experience. I an about to attempt a 
> protocol for preservation of lipid for paraffin 
> sectioning as per
> 
> Virchows Arch (2004) 445:22-26
> Richard E. Tracy  Parvene Walia
> Lipid fixation for fat staining in paraffin sections applied
> to lesions of atherosclerosis
> 
> and
> 
> A method to fix lipids for staining fat embolism
> in paraffin sections
> R E Tracy & P Walia
> Histopathology 2002, 41, 75-79
> 
> My question is whether a day at 2% chromic acid 
> (24 h at 4 degrees C) is likely to decalcify the 
> soft tissues? We are trying to look at 
> atherosclerotic lesions including lipids (Oil Red 
> O) and calcium deposits (von Kossa stain)  in 
> heart, liver, and kidney. Has anyone been able to 
> perform RNA in situ or IHC on these samples after 
> this type of processing? We do not have access to 
> a cryostat. If anyone has tried this protocol 
> with (or without) success, I'd be grateful for 
> the advice.
> 
> Thanks in advance,
> Nicole Collette
> 
> LLNL/UCB
> collette2@llnl.gov
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