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Re: [Chlamydomonas] Chlamy Fluorescent Immunostaining

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Hi Heloisa,

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:

http://chlamy.tiddlyspot.com/#%5B%5BIF%20Staining%20-%20Chlamy%5D%5D


Good luck,
Ben

On Mon, Jan 25, 2016 at 2:09 PM, Heloisa Ciol <helociol@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
> Chlamy@net.bio.net
> http://www.bio.net/biomail/listinfo/chlamy
>

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Re: [Chlamydomonas] Chlamy Fluorescent Immunostaining Ben Engel <bdengel@gmail.com> - 2016-01-25 17:03 +0100

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