Chlamy Fluorescent Immunostaining

[Heloisa Ciol]

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

[Ben Engel]

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

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[Ben Engel]

Hi Heloisa,

Your cells should stick. My guess is that you aren't getting a good poly-L
coating of the coverslip. Maybe your poly-L is bad or at too low of a
concentration. If I remember correctly, we used poly-L that is 10x more
concentrated than what is used for mammalian cell culture.

I think this 0.1% solution from Sigma might be the one. It should just
come in a big plastic bottle:
http://www.sigmaaldrich.com/catalog/product/sigma/p8920

If you are only using 0.01%, that is likely your problem. Also make sure
that your culture is fairly dense, at least medium green or even a bit dark
green.

Good luck,
Ben



On Mon, Jan 25, 2016 at 8:20 PM, Heloisa Ciol <helo...@gmail.com> wrote:

> Hi Ben and Telsa,
>
> Thank you for sharing your protocols and experience. I'm sorry, I forgot
> to mention: As we didn't succeed in sticking cells on the coverslips, we
> used glutaraldehyde to fix the cells. I tried to use methanol in one of my
> trials, but I couldn't find a single cell at the coverslip at the end of
> the protocol.
>
> I will try Ben's protocol this week in hope to succeed and let you know
> once I checked the staining at the confocal.
>
> Thank you all one more time.
>
> Bests,
>
> 2016-01-25 15:40 GMT-02:00 Mittelmeier, Telsa M - (telsa) <
> te...@email.arizona.edu>:
>
>> Hi Heloisa,
>>
>> Attached is the protocol that I have been using for dual immunostaining
>> with anti-acetylated tubulin (mouse monoclonal) plus a rabbit polyclonal
>> directed against a particular eyespot protein. The cell suspension that I
>> put on the coverslips is medium green, and I would guess that about 10% -
>> 25% of the cells end up sticking to the untreated coverslips...plenty for
>> imaging.
>>
>> Sometimes the IF looks great, other times, not so great. I have a couple
>> of problems that I am not sure how to solve (I have cc'd Ben for any
>> suggestions?): sometimes large numbers of the flagella fall off, and
>> sometimes I get "schleering" or heavy background from the Mowiol (I
>> think)...I have not yet found the perfect method for coverslipping.
>>
>> Hope this helps!
>>
>> Sincerely,
>>
>> Telsa Mittelmeier
>>
>> (Carol Dieckmann's lab, Univ of Arizona...we study the Chlamy eyespot)
>> ________________________________________
>> From: chlamy-...@oat.bio.indiana.edu <
>> chlamy-...@oat.bio.indiana.edu> on behalf of Ben Engel <
>> bde...@gmail.com>
>> Sent: Monday, January 25, 2016 9:03 AM
>> To: Heloisa Ciol
>> Cc: chl...@magpie.bio.indiana.edu
>> Subject: Re: [Chlamydomonas] Chlamy Fluorescent Immunostaining

> --
> Heloisa Ciol
>