I have to do, for the first time, immunofluorescence on cells in suspension and just I don't know how to proceed.
Hi,
I may have a protocol for you.
The main difference to normal slide immuno essays usually is on the way you do the washes, I perform a series of low speed spin-down´s to recover cells with out compromising cell integrity.
But in order to give you a accurate answer, I need more information... what is the cell type (bacteria, plant cells ....), do you want to do the assay on live or fixated material?..
Mário
Please feel free to contact me at [email protected]
Thank you Mario for your availability!
My cells are rat cells from testicole.
They grow attached to the culture plate and normally I do immunofluorescence "in-situ", but for new experiments I need to do it detaching them.
My question is also about fixation or not-fixation....
When I have to do it and when not? Which is the best way to fix the cells?
Thankks for any help you can give me Mario!
Alessandra
ok, so you have animal cells.. I work with plant cells (pollen tube) that are a lot more resistant. (so spin down speeds and buffers molarities on my protocol, will probably be wrong for your experiment..)
So lets get this one part at the time..
You can do the hybridization pretty much in the same way that you normally do, except that you make it on 2ml eppendorfs and the washing steps, every time you change a solution you do a spin down (5 000 g 6 min in At pollen tubes) discard supernatant and resuspend the pellet in the next solution.
About fixation... it depends on what you are looking to observe if its insitu hibridization for RNA you should fix the cells... if its proteins you should probably also fix the cells you should consider always to fix the cells unless you are looking for a change that may occur during the immunolocalization.
Hope it helps
Mário
You can spread cells or make smear preparation on PLL coated glass slides.
See: Immunohistochemical localization of mitochondrial fatty acid β-oxidation enzymes in Müller cells of the retina.Atsuzawa K, Nakazawa A, Mizutani K, Fukasawa M, Yamamoto N, Hashimoto T, Usuda N.Histochem Cell Biol. 2010 Dec;134(6):565-79.
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