From owner-celegans@net.bio.net Mon Mar 24 22:00:00 1997 Path: biosci!agate!spool.mu.edu!howland.erols.net!newsxfer.itd.umich.edu!yale!oitnews.harvard.edu!hpngsv01.mgh.harvard.edu!atgw-wel-800d-1-c12.mgh.harvard.edu!user From: patterson@frodo.mgh.harvard.edu (Garth Patterson) Newsgroups: bionet.celegans Subject: Re: visualizing mitotic spindle Date: Mon, 24 Mar 1997 14:11:16 -0500 Organization: Molec. Biol., Mass. General Hospital Lines: 76 Message-ID: References: NNTP-Posting-Host: atgw-wel-800d-1-c12.mgh.harvard.edu I got several replies to my post regarding visualizing spindles, which are reproduced below. Thanks to all From: Monique Kreutzer 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@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 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" 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@frodo.mgh.harvard.edu .