Worm Breeder's Gazette 8(3): 76

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Localization of Class C Acetylcholinesterase by Immunocytochemistry

B. Stern

I have partially purified class C acetylcholinesterase (AChE) from 
triton extracts of N2 animals and prepared polyclonal antibodies to 
the partially purified protein.  A rabbit was immunized with a band 
cut out of an SDS gel, boosted once with another cut out band and then 
boosted twice more with  undenatured samples.  I estimate that each 
injection contained about 10 g of enzyme.  Anti-class C antibodies 
were detected only after the third boost.  By ELISA assay the 
antibodies show no cross-reactivity to forms from the other two known 
AChE classes, A and B.  However, by immulunoblots the antibodies did 
show cross-reactivity to a number of other C.  elegans proteins.  
Absorption of the antibodies with an extract from ace-3(dc2) animals 
resulted in the removal of these contaminating antibodies, as shown by 
immunoblots.  (Animals containing the dc2 allele have no detectable 
class C enzymatic activity and by ELISA have no cross-reactive 
material, and therefore are apparently null.)  I have used the 
following procedure to determine the localization of class C in whole 
1.  Densely settled animals are squashed on 1.0% BSA subbed slides 
according to the procedure of Albertson et al (WBG 7(1):73).  I find 
that squashing with a cover slip is most convenient.  After freezing 
in liquid nitrogen and prying off the cover slip with a razor blade 
the slides are passed through methanol, acetone, and a graded series 
of ethanols to TBS (Tris buffered saline).  The following incubations 
are then performed in a humid chamber, with 0.035 ml reagent placed 
over the squashed animals.  
2.  Incubation with 10% normal goat serum (NGS) in TBS for 30 
minutes, followed by a brief wash in TBS.  
3.  Incubation with primary antibody (1:1000) diluted in 1% NGS/TBS 
overnight, followed by two 10 minute washes in TBS.  
4.  Incubation with Goat anti-Rabbit IgG antiserum (1:150) in 1% 
NGS/TBS 30 minutes, followed by a brief wash in TBS.  
5.  Incubation with PAP reagent (1:100) in 1% NGS/TBS 30 minutes, 
followed by 2 ten minute washes in TBS.  
6.  Repeat the two previous steps.  
7.  Develop the peroxidase reaction with 0.44 mg/ml diaminobenzidine 
HCl 0.003% H202 in TBS for 5-10 minutes, followed by a brief wash in 
8.  Slides are then mounted with 90% glycerin in TBS.
The brown reaction product is visible in the nerve ring, in several 
cell bodies near the nerve ring, and in two cells located near the 
vulva.  In addition to the specific staining seen in these structures 
there is a diffuse background which is visible.  When staining dc2 
there is no specific staining, however the background persists.  Some 
non-specific staining is also observed in the pharyngeo-intestinal 
valve, both in N2 and dc2.  The two cell bodies are located under the 
lateral alae as are their processes which stain and run posteriorly to 
the level of the anus and anteriorly to the level of the pharyngeo-
intestinal valve.  I believe that these are the CAN cells.  They stain 
quite well in late stage embryos and L1s, as well as older animals, 
and are Present in unc-86 animals.  As to why the CAN cells should 
have class C AChE on their surfaces is anyone's guess.  While these 
cells are required for viability as shown by John Sulston, that 
viability is not dependent upon class C AChE since the ace-3 animals 
are viable, and in fact show no visible phenotype.  
In addition to the above mentioned staining I also occasionally see 
staining in cells which I believe are the PDE cells, based on their 
location and morphology.  Work is in progress to confirm this result 
as well as to stain animals with altered CAN cells (vab-8).