Worm Breeder's Gazette 11(2): 20a

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Title unknown.

Authors unknown.

Various protocols for permeabilization and staining of adult C.  
elegans in such a way as to avoid killing embryos in the uterus have 
been described (Sebastiano et al Genetics 112 459, AF WBG 9#2, Stone 
and Shaw WBG 10#3 p27, Candido MRC lore).  I have been trying to 
optimize this procedure to obtain strong, well localized staining of 
lines carrying  -gal fusion proteins, to allow for selection of 
altered staining patterns after mutagenesis.  A current protocol is 
described below.  The treatment is somewhat more drastic than previous 
methods, but allows excellent staining and reasonable preservation 
with survival of 3-5 embryos per stained adult.
1.  Pellet animals from H20 and resuspend in 50% acetone for 10-15 
min at room temp (the time for this step is critical...too long and 
embryos are inviable, too short and the adults fail to permeabilize 
and awaken in the staining solution.).  2.  Wash animals 3x in PMB and 
resuspend in staining mix.  Stain at room temperature.  The hatched 
animals can survive within the adult in staining solution for several 
hours, but eventually die or free themselves, limiting staining time 
to about 12 hr.  3.  Scoring with a dissecting microscope is best with 
epi-illumination over a white 
background
PMB: 50mM NaPi pH 7.5, 1mM 
MgCl2
Staining Mix: PMB + 10mM KFerrocyanide + 10mM KFerricyanide + .004% 
SDS + 10  l/ml of 12.5% XGAL in DMF (XGAL stock should be stored at -
70 C)
Optimal permeabilization so far has been around 95% of animals.  
Thus the technique could be used to screen for ectopic expression or 
expression of an otherwise inactive construct, but loss of expression 
would be difficult to screen for.
N.B.  Some permeabilization of pharyngeal muscle occurs without the 
acetone step (putting animals directly into staining solution).