From owner-celegans@net.bio.net Tue Nov 19 22:00:00 1996 Path: biosci!JEEVES.UCSD.EDU!trakow From: trakow@JEEVES.UCSD.EDU (Terese Rakow) Newsgroups: bionet.celegans Subject: antibody controls Date: 20 Nov 1996 08:10:45 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 88 Sender: daemon@net.bio.net Distribution: world Message-ID: NNTP-Posting-Host: net.bio.net I've had some requests to post the answers I received to my qusetion about use of control antibodies for whole worm staining. Thanks to all who responded. We are muddling through the many protocols out there. What I've gathered is that there is a different control that works for each (maybe not exclusively) of the many protocols. Terese Rakow >>>>>>>>>>> MH27 is an excellent control for staining. It is a monoclonal, specific for adherens belt junctions at apical junctions between hypodermal cells and between intestinal cells. It therefore has an easily recognizable, specific pattern of staining. Because it is a monoclonal, it also is fairly sensitive to your fixation conditions, and could therefore act as a good control for your protocol (the possibly faulty reasoning being that if it works for MH27, it should work for other antibodies). I could send you some supernatent if you like. It has a pretty high concentration of antibody, and I have tested it recently. You would only need about 1 ul per 50 ul of fixed worms. You need to stain secondarily with rhodamine- or fluorescein-conjugated goat anti-mouse IgG (I recommend rhodamine for better resistance to photobleaching). David Fitch Terese, You can try tubulin (DM1a from SIGMA) or actin (C4 from ICN). I don't know what stages you are looking at or which tissues so these may or may not be appropriate. Good luck and see ya!! Raffi The ATCC (American Tissue Culture Center) sells sells monoclonal antibodies that stain different tissues in C. elegans (the monoclonal cell lines were made at the MRC). These are 3NB12 (to pharynx), 1CB4 (to gut), NE2/1B4.14 (to seam), and NE8/4B6.3 (to muscle). You could also use anti-betagalactosidase antibody from Promega (at 1:1000 dilution) to stain any line with a lacZ reporter gene. All of these work well with methanol/acetone fixation (and others), but some antigens need other fixation methods. Bruce Bowerman told me that SKN-1 requires paraformaldehyde/methanol fixation, and this is the only fixation where I can see anti-VAB-7 staining. You could write him for a protocol. Julie Ahringer Steve Salser (Kenyon lab) used a histone antibody for anti-mab-5 controls to test permeabilization, although I'm not sure where he got it. Unfortunately, fixation protocols really need to be optimized for each individual antibody/epitope combination. The histone ab will only tell you whether that particular epitope is preserved and the worm is sufficiently permeable to allow abs in. Another radically different fixation technique - methanol/acetone with frozen cracking - can be tested with the MH27 ab as a control. Curtis Loer GFP monoclonal and polyclonal antibodies are available from Clontech, and could be used with any GFP transgenic you have. Garth I assume you are staining ce. A good positive ab would be one which stains the nervous system. We have been using one which labels over 50 percent of the NS-- however, what are you trying to stain. David Brownlee Hi Terese - I think you were asking about good control antibodies. There are several that I have used - probably the most versatile in terms of fixations is an anti-DNA antibody called mab030 from Chemicon. It works on paraformaldehyde and MeOH/Acetone fixed worms and it stains all nuclei. I have also used an anti-actin antibody called C4 from ICN and an anti-B-tubulin from AMersham. The anti-actin and anti-tubulin work best on MeOH/Acetone fixed worms or gonads that have been extracted with MeOH before fixation with paraformaldehyde. Hope this helps! Sarah Crittenden Yoy could use an antibody against serotonin if you are fixing with just paraformaldehyde. If you are fixing with both glutaraldehyde and paraformaldehyde you could use the antibody against GABA. Incstar apparently has a reasonable GABA antibody, I imagine their serotonin antibody is also acceptable at least as a control. We would look at the animals in collagenase with a dissecting scope and terminate the digestion after 10-30% of the animals had basically broken apart. Let me know if I can be of further help. Steve McIntire Terese Rakow Dept. Biology-0116 5165 Muir Biology Bldg. UCSD 9500 Gilman Dr. La Jolla CA 92093-0116 .