I was wondering if there is a reason to use one method over the other, depending on what you're looking and staining for or depending on the cell type?
From what I know, methanol denatures and precipitates proteins as well as makes the cell membranes permeable, where formaldehyde crosslinks proteins but does not permeable well, so something like Triton has to be added afterwards.
I'm staining for a nuclear protein in developing neural cells derived from stem cells.
Hello Alex,
in General, it's depend for what you looking for.
Formalin is mostyl used in the Routine histology. Methanol for more specific diagnostic like cytodiagnosis.
Methanol has the Advantage the it remove the lipids in the tissue. But it's toxic. So.. just try both way and look what give you in These Special test the best results.
Cheers Robert
They are two different fixatives.
Methanol is an alcohol which dehydrate cells instantly. Many lipids are removed from membranes, proteins precipitate. Small soluble metabolites leak and cells shrink.
It is suitable for immuno as it keeps intact epitopes, for nucleic acid protocols such as ISH, FISH, caryotype, or tanscriptomics, but all structures in the cell are modified, collapsed.
On other hand Formaldehyde (paraformaldehyde if you prepare it from powder, formalin if you buy it in 36% solution) is an aldehyde, which mildly oxidize reduced carbon or nitrogen. It creates bonds between proteins, lipids or between lipids and proteins.
Cell structures are maintained, membranes remain intact (but permeable) but epitopes might be modified, nucleic acids are modified and a general, non specific can be produced (highly problematic in plant cells).
As this FA fixation is rather slow, proteins can move through the cell before being fixed, so in certain circumstances, you can see nuclear protein in the cytosol, or membranary proteins in the ER.
For your purpose I think formaldehyde fixation would be most suitable as the cell-type you're working on is flat and transparent with a very specific shape you may want to maintain.
Both formaldehyde and methanol are toxic products. Methanol is neurotoxic that can lead to blindness, while formaldehyde is a known CMR causing mutations (lung cancer or male sterility), so take all the needed precautions.
Hello Alex,
If your cells are a little sensitive, another option is to try to fix with 10% neutral buffered formalin for 15 minutes rather than the 4% PFA. The cell morphology is preserved well. Permeabilize the cells with 0.5% tween for 20 minutes. I have had a lot of success with this method when staining primary cell lines for IF.
maybe this link will be helpful...
https://blog.cellsignal.com/successful-immunofluorescence-fixation-and-permeabilization
I want to perform ICC/IF on the shsy5y cells, human neuroblastoma, but a bit confused with the type of fixation :
1) should I fix with the 100% methanol for 1 hr?
2) 4% paraformaldehyde for 10 minutes?
Elah Pick
Fixation by alcohols works by removing the hydrate cover, causing the proteins to collapse and re-fold in the process, rendering them insoluble. Methanol is merely the fastest penetrating, but you can achieve similar (but worse) results with ethanol, acetone, chloroform, etc.
The proteins are only structurally, but (mostly) not chemically altered, so they should retain their antigeneticity. If you need high specificity and label density, and/or low background fluorescence but can afford structural degeneration or sunbstance loss, this is your way.
Aldehyde fixation introduces crosslinks between proteins, more precisely between functional groups of amino acid sidechains in close proximity, both between different positions of the same amino acid chain and neighboring amino acid chains. In the process, proteins are rendered insoluble, but also are chemically altered, which might affect antigeneticity. But they are generally mechanically more stable and better suited for subsequent processing like resin embedding. Aldehydes introduced into the tissue may exhibit autofluorescence, though.
If you can afford some loss in label density and specificity and/or autofluorescence, but need good structural preservation and/or have to prevent loss of cell content during subsequent processing, this is your way to go.
It really depends on what you want to do with your fixed cells afterwards. There is no artefact-free chemical fixation, you need to choose a method the artefacts introduced by which don´t bother you further.
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