Hoechst assay - cuticular permeability or rupture?

I’ve got the Hoechst cuticular permeability working quite well with clear nuclear staining in my bus-8 mutant and nothing,
apart from gut fluorescence, in N2. My mutant strain exhibits acute hypo-osmotic sensitivity and is essentially equivalent
in this respect to the CB1072 unc-29 (non-alpha subunit of nAChR) strain. The bus-8 mutant exhibits clear nuclear staining
that can be captured by the camera at relatively low exposure times. Some, but not all, CB1072 and my strain also exhibit
nuclear staining but it is much fainter requiring longer exposures to view nuclei. Even though I’m using M9 containing
0.1% gelatin which appears to attenuate ‘popping’ on the slide, these worms still become very stiff and occasionally burst.
Popped worms stain v easily presumably because the dye has physical entry to the worm. So what if there’s a very minor,
non-visible rupture of the cuticle in some non-popped worms that allows dye entry and thus staining creating, essentially,
a false positive for cuticle permeability? Could this be causing the faint staining I see in some CB10072 and my strain?

have you tried doing the same test using an isotonic solution in place of M9?

If you leave the worms in 0.1% gelatin for longer time periods will they all eventually pop? A non-visible cuticle rupture could cause the staining, but this may be hard to test unless you have access to TEM or SEM. Only other thing that I can think of is maybe cross in a COL-19:GFP marker to see if there are any obvious cuticle defects, like alae gaps or problems with the annuli. One other question is are you doing mixed stage staining or staged staining? I believe that the dye may have faciliated access during the molt, so you might want to see if there are any developmental progression or molting phenotypes.