What type of fixation/permeabilization are you using? Cold methanol is
generally best for staining microtubules (but it is bad for other
structures like actin). It also extracts the chlorophyll, which is a plus
for immunofluorescence. Back when I used to stain chlamy cells, I put a
drop of dense culture on the poly-L coverslip, let the cells adhere for a
few minutes, drained off the excess, and then immediately put the
coverslips in a coplin jar of cold methanol in the freezer. Of course we
lost lots of cells, but there were always plenty on the slide.
Here is an old protocol that I used when I was in the Marshall lab. It
definitely works very well for acetylated tubulin. So if you try this and
don't see anything, I would blame your antibody. I have no idea anymore
what is the best acetylated tubulin antibody for Chlamy, but I'm sure
someone else on this list will know:
On Mon, Jan 25, 2016 at 2:09 PM, Heloisa Ciol <helociol from gmail.com> wrote:
> Dear all,
>> I was wondering if anyone could share here some experience in Chlamy
> fluorescent immunostaining, please?
>> Since I started in this field, I couldn't apply to my experiments any of
> the protocols mostly used so far. For some reason, I cannot fix my cells in
> coverslips (I prepared coverslips at the lab, either using poly-L-lysine or
> polyethyleneimine). At first the cells seem to be stuck at the glass, but
> once I go to the washing steps, almost all of them disappear, and in the
> end, I can find 2- 5 cells at the whole coverslip. To overcome that, we
> developed a protocol for cell incubation in eppendorfs with blocking and
> antibody solutions, which gives us a lot of cells, but not an uniform
> staining pattern.
>> Besides that, we are trying to use alpha-tubulin or
> alpha-acetylated-tubulin commercial antibodies as well, but we couldn't
> stain a single cell so far.
>> Does anyone have a tip or a protocol to share that I could try using here
> in our lab?
>> Thank you all.
>> Best regards,
> Heloisa Ciol
> Chlamy mailing list
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