Can anyone share some worm immunohistochemistry experience?

I am trying to detect the expression of a transgene protein in the neuron. I used Bouin’s fix solution and BTB (Boric acid, triton x 100,mercaptoethanol) to fix and permeabilize the worms, I froze and thawed 3 times with dry ice and ethanol,
I could not get the adult worms stained, only the very young worms show positive.
Is it because that the adult worms have a thick cuticle? I am considering using collagenase, will collagenase treatment help?

Thanks in advance!

I do recommend trying collagenase to help permeabilize the worms. This is especially true if you are fixing for a long period of time. hope you are having more success.

Thank you, Janet, how long is called “long period” ? overnight?

I would consider more than an hour to be a “long period” - overnight certainly counts as long.
I recommend that you either try a brief fixation (15-30 minutes) or collagenase after a longer fixation (e.g., overnight)