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Fixing tissue


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#1 Helios

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Posted 10 November 2009 - 09:11 PM

Has anyone tried immunohistochemistry on non-fixed tissue samples????
also which fixative in your experience is the best to be used for IHC????

#2 skeuos

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Posted 11 November 2009 - 11:10 AM

Has anyone tried immunohistochemistry on non-fixed tissue samples????
also which fixative in your experience is the best to be used for IHC????




A lot of histochemistry techniques I've used in the past rely on non-fixed tissues; I then used those tissues for immunohistochemistry without problem. I did fix them on the slide, usually with cold acetone. Other alternatives include cold methanol, NBF, or PFA to name a few. I think I've used them all at one point or another. The fixative you choose depends on what you need - permeabilizing the cells, avoiding autofluorescence, or preserving GFP, etc.

Are you asking about fixing the tissues on the slide, or a fixation beore embedding?

#3 bob1

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Posted 11 November 2009 - 04:47 PM

usually with cold acetone. Other alternatives include cold methanol, NBF, or PFA to name a few.

All of which are fixatives...

Has anyone tried immunohistochemistry on non-fixed tissue samples????
also which fixative in your experience is the best to be used for IHC????

As skeuos says the fixative you use depends on what you want to do. I like PFA myself as it preserves structure and shape of cells really well, but it is slow and unstable. Methanol and acetone are good but dehydrate the cells so they lose their shape.

#4 Helios

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Posted 16 November 2009 - 10:49 PM

thanks for the answers....i was asking about fixing the sections on slides before the staiining procedure.

#5 bob1

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Posted 17 November 2009 - 03:50 PM

To attach cultured cells to the slide? Try gelatin (collagen), or poly L-lysine, they work for most cells.

To attach tissue chunks/slices to slides, most people dry them on, but you can use things like APES (3-Aminopropyltriethoxysilane) coated slides.

#6 SuMi

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Posted 24 November 2009 - 03:03 AM

Just to add to this. If a tissue is fixed in formalin for 24 hours, can you perform histochemical staining without going through the paraffin-embedding stage? i.e. if i leave the samples in ethanol for 48 hours and then PBS for 48 hours can I then stain them? I'm having trouble figuring out a way of preserving whole tissues since I don't want to section them.

#7 bob1

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Posted 24 November 2009 - 03:37 PM

Just to add to this. If a tissue is fixed in formalin for 24 hours, can you perform histochemical staining without going through the paraffin-embedding stage? i.e. if i leave the samples in ethanol for 48 hours and then PBS for 48 hours can I then stain them? I'm having trouble figuring out a way of preserving whole tissues since I don't want to section them.

Absolutely. Check out whole mount embryo protocols for techniques. The biggest issue is getting the antibodies into the tissue with lumps. You may have to look at getting some Fab fragments to get around this problem. Also make sure you wash out the formalin really well, as the aldehydes formed during the fixing process interfere with antibody binding. Lots of long washes in PBS should do it, but you can also try 0.1 M glycine in PBS.

#8 SuMi

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Posted 25 November 2009 - 01:24 AM

That's brilliant thanks. I'm not staining for antibodies, it's just a lectin stain so I don't think I will have a problem with antigen retrieval. Thanks again




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