Can anyone tell me, what is the principle behind cell fixation before any staining and also what happens to cell when we are treating it with the fixing agents like methanol, para-formaldehyde etc?
The mechanism of fixation is dependent on the reagent used. Alcohol based fixations dehydrate cells/tissues, causing proteins to denature and precipitate in situ. Paraformaldehyde causes covalent cross-links between molecules, effectively gluing them together into an insoluble meshwork.
The reason cells must be fixed prior to immunostaining is quite simple. You need to permeabilize cells to allow antibodies to access intracellular structures. Without fixation, the structures in cells would fall apart and diffuse away before you had a chance to finish the antibody incubations and wash steps.
Good start to read is this one: http://www.leicabiosystems.com/pathologyleaders/fixation-and-fixatives-1-the-process-of-fixation-and-the-nature-of-fixatives/
The mechanism of fixation is dependent on the reagent used. Alcohol based fixations dehydrate cells/tissues, causing proteins to denature and precipitate in situ. Paraformaldehyde causes covalent cross-links between molecules, effectively gluing them together into an insoluble meshwork.
The reason cells must be fixed prior to immunostaining is quite simple. You need to permeabilize cells to allow antibodies to access intracellular structures. Without fixation, the structures in cells would fall apart and diffuse away before you had a chance to finish the antibody incubations and wash steps.
Hi Raj
In addition to the above, The application of the different types of fixatives may takes place by immersion or by perfusion (injection). The latter method is widely used in immunohistochemistry specially the nervous tissues. The selection of the type of the fixative depends on the tissue staining method and the tissue demonstration (routine histology, histochemistry, immunohistochemistry or cytological organelles). The removal of the fixative traces, after fixation, from tissue must be carried out because it impede the process of proper staining. The amount of the fixative used should be at least twenty to thirty times the volume of the specimen. The specimen should be small to allow good penetration of the fixative used. Finally, the time of fixation depends upon the size and type of the specimen as well as the type and volume of the fixative used.
Best regards
I would also like to add that for protocols like immunocytochemical staining, fixation via most methods will also preserve the structure of protein cellular scaffold. This is important as when cells dehydrate or degrade overtime, protein, membrane and intracellular structure will also alter or degrade. This can then prevent proper detection with antibodies or stains. Fixation with alcohols such as methanol has the added advantage that it can also permeabilise the cell, whereas fixation with paraformaldehyde leaves the membrane intact so an additional membrane permeabilisation step is required.
fixation is the main step in histological and the field like that to study on the slide of tissue, cells & ets. as a histologist you shoud know the healthy tissue formation and structure to recognize the tissue problems and the unhealthy one.
as hematologist you should recognize the blood cells size, colours and ets.
briefly i want to say fixation is needed because it keeps the tissue, cells and... in real shape and charactristics. you need to study on the cells or tissue but before study, you shoul prepare the fram which you study on it and fixation is the main step that keep the sample in shape.
hi,
The mechanism of fixation is dependent on the reagent used. Paraformaldehyde causes covalent cross-links between molecules, effectively gluing them together into an insoluble meshwork. i would like to attach my protocol here.
is fixation done in a solution state? what i mean is if I trypsinize cardiac cells off the petri dish ,then can I still add PFA to it. I actually tried doing that but somehow after 10 minutes incubation all the cells were digested and i got absolute no pellet post centrifugation.
Please suggest.
Thanks
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