Hi All,
Does anyone have a good protocol for staining bacterial cells (in suspension culture) with a primary Ab followed by a fluorescently-conjugated secondary Ab? I'm just wondering primarily how to get the cells on the slide, fix them (if required), and what types of washes are good. The protein I'm trying to label is extracellular so I don't think a permeabilzation step is needed.
Thanks a lot,
Adam
Hi Adam
Fixing and staining bacteria in suspension is fairly straight-forward.
1. Wash cells in, e.g., TBS
2. Resuspend cells in 3.7% formaldehyde for 5 minutes
3. Wash cells twice in TBS to remove the formaldehyde
4. Resuspend cells in AbDil contining your primary antibody, anywhere from 1:500 to 1:5000 antibody dilution usually, for 1 hour (see http://mitchison.med.harvard.edu/protocols/general/Fluorescence%20Procedures%20for%20the%20Actin%20and%20Tubulin%20Cytoskeleton%20in%20Fixed%20Cells.pdf for the recipe)
5. Wash cells 3 times to remove the primary antibody. You can use TBS-Tx at 0.02% maybe? Might get a better wash, but I guess it depends on how your protein of interest is anchored.
6. Repeat steps 4 and 5 for your secondary antibody. We use a bit more secondary, usually only a 1:200 to 1:500 dilution, and only incubate for about 40 minutes. This could be over-kill though.
7. If you want to use DAPI then switch to plain TBS from now as detergents and DAPI are not good friends. Incubate TBS-DAPI (1ug/ml) with the cells for 5 minutes and wash in TBS.
8. Spot your cells onto coverslips coated in either Concanavalin A or Poly-L-lysine and let them attach, but not dry out completely. Poly-lysine might give you some autofluorescence in green (I have not seen this, but others have mentioned it).
9. Mount the coverslips onto slides using your favourite mounting medium and seal with VALAP/nail varnish. I strongly recommend making the medium detailed in the PDF above.
You might need to tweak this for your own purposes, but hopefully it's a good place to start. Have fun on the microscope!!
Hi Michael,
Thanks for the protocol, this really helps a lot!
Best regards,
Adam
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