visualizing mitotic spindle

Garth Patterson patterson at frodo.mgh.harvard.edu
Mon Mar 24 14:11:16 EST 1997


I got several replies to my post regarding visualizing spindles, which are
reproduced below.  Thanks to all


From: Monique Kreutzer <kreutzer at helix.mgh.harvard.edu>

Using N357 (Amersham), anti-beta tubulin, I primary detect the staining of
the centrosomes but also get some staining of the spindles themselves.  The
anti-alpha tubulin antibody N356 (also from Amersham) stains the entire
spindle apparatus much better in my hands.

The N357 anti-beta tubulin antibodies works very well in my hands either
using methanol/acetone fixation on embryos or the Finney or Bowin
protocols(from the latest edition of the worm breeder's gazette) on larvae.
If you would like more details concerning the fixations just let me know.
I can get very adequate staining using a 1:50 to 1:100 dilution of N357
however, 1:25 gives photo quality staining.  I dilute the primary in 1%
BSA/TBS +10%Normal Goat Serum.  The secondary I use is goat anti-mouse FITC
conjugated that was purchased from Cappel, Catalog number 55493.  The
dilution I use is 1:50 to 1:100.  The dilution solution is 1%BSA/TBS +10%
NGS.  The incubation conditions for the primary and secondary antibodies
vary depending on the protocols but based on the protocols themselves it
works best with an overnight incubation with the primary antibody.  The
secondary antibody incubation I do for 2 hours at room temperature or 4
hours in the refrigerator/cold room.

The N356 antibodies work very well using methanol/acetone fixation on
embryos and I have never tried it on larvae.  I get very good staining of
the entire spindle apparatus using 1:150 to 1:300 dilutions.  I use the
same secondary as described for N357. 

------------------
From: david.greenstein at mcmail.vanderbilt.edu

For our studies in germline development, we find that two antibodies are
particularly useful for detecting meiotic and mitotic spindles in the gonad and
early embryos.  We find that fixation with methanol acetone gives the best
detection in these preparations.

1)  Monoclonal anti-b-tubulin N357  from Amersham--a mouse monoclonal

2)  YL1/2 anti-alpha tubulin--a famous Rat monoclonal originally described by
Kilmartin et al., JCB 93: 576-582, 1982.  Obtained (a long time ago) from Sera-
Lab.


Sometimes we even mix the two. I have never tried to detect the spindles in
intestinal cells. 
--------------------

From: Dominique Bergmann <Dominique.Bergmann at Colorado.EDU>

The Piperno and Fuller monoclonal antibodies to alpha-tubulin (3A5)
stain the spindles of early embryos beautifully.  I haven't looked at 
later stages, but they might be worth a try.
-------------------
From: "Neville R. Ashcroft" <ashcrofn at nciaxp.ncifcrf.gov>

A 1984 method by Donna Albertson suggests using antitubulin YL1/2 or
YOL1/34 antibodies (Dev Biol101, 61-72).


> I would like to be able to visualize the mitotic spindle in dividing
> intestinal nuclei (at the end of L1).  I guess antibodies to a component
> of the microtubules would be ideal.  Does anyone know where I could get
> such antibodies, or does anyone have an alternative suggestion?
> 
> Thanks,
> 
> Garth

-- 
Garth Patterson
Dept. of Molecular Biology, Mass. General Hospital
Dept. of Genetics, Harvard Medical School
patterson at frodo.mgh.harvard.edu



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