trakow at JEEVES.UCSD.EDU
Wed Nov 20 11:10:45 EST 1996
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.
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).
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.
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
GFP monoclonal and polyclonal antibodies are available from Clontech, and
could be used with any GFP transgenic you have.
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
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
5165 Muir Biology Bldg.
UCSD 9500 Gilman Dr.
La Jolla CA 92093-0116
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