[Histonet] time for penetration of methacarn fixative
jkiernan at uwo.ca
Sun May 5 23:26:24 CDT 2019
Peter Noyce, you did not say what tissue(s) you are planning to fix, or how big the specimens will be.
Carnoy (1886) and methacarn (1970) were developed for animal tissues, cleverly balancing the actions of acetic acid and an alcohol in the presence of a hydrophobic solvent (chloroform) that diluted both these ingredients and also enhanced permeation by dissolving lipids. Carnoy's and other acid-alcohol fixatives are also used for plant specimens, which respond differently. If you are working with plant specimens you probably have a book by Steven E. Ruzin (1999) ISBN 0195089561.
For the actions of alcohols and acetic acid in fixation of animal tissues, consult any text book of histotechnology or histochemistry published since about 1950.
For selfish reasons, I recommend ISBN 9781907904325 (published in 2015) as an item to buy for your lab's bookshelf. Answers to your question are discussed in Chapter 5.
With methacarn and other alcohol-acetic fixatives, no slow chemical reactions are involved (an important difference from formaldehyde-containing mixtures). Complete penetration accomplishes the fixation. The volume of fixative and procedure for subsequent processing into paraffin are very important. You need to read the paper by Puchtler et al (1970) and follow the instructions exactly. Probably you should also read Puchtler et al (1968) to use this type of non-aqueous fixative intelligently. PhD students are intelligent.
I have added a couple of more recent papers that relate to methacarn. Read Puchtler or a textbook first.
Not all modern investigators (especially molecular biologists) understand what the ingredients of fixative mixtures do to the different components of cells and extracellular materials. Current papers with micrographs full of ghastly artifacts abound, even in journals with very high citation indices. There are published mixtures with names like "modified methacarn" that may be OK for extracting RNA but do not have ingredients in correct proportions for minimizing distortion.
Here's your list of recommended readings.
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Puchtler H, Waldrop FS, Meloan SN, Terry MS, Connor HM (1970) Methacarn (methanol-Carnoy) fixation. Practical and theoretical considerations. Histochemie 21: 97-116.
Puchtler H, Waldrop FS, Conner HM, Terry MS (1968) Carnoy fixation: practical and theoretical considerations. Histochemie 16: 361-371.
Uneyama C, Shibutani M, Masutomi N, Takagi H, Hirose M (2002) Methacarn fixation for genomic DNA analysis in microdissected paraffin-embedded tissue specimens. J. Histochem. Cytochem. 50: 1237-1245.
Buesa RJ (2008) Histology without formalin? Ann. Diagn. Path. 12: 387-396.
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I wish you well with your research, and hope you will get your PhD while you are still young.
= = =
From: peter noyce via Histonet <histonet at lists.utsouthwestern.edu>
Sent: 03 May 2019 19:47
To: histonet at lists.utsouthwestern.edu
Subject: [Histonet] time for penetration of methacarn fixative
Does any one have data for the time it takes for methacarn fixative (60%
methanol, 30% chloroform, 10% glacial acetic acid) to penetrate and then
fully fix tissue?
PW Noyce -PhD student
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