suitable fixation protocol for pharynx staining

Hi all,

I use 4% paraformaldehyde+5% methanol as a fixative to fix my worms using the Modified Finney-Ruvkun protocol. My aim is to use these fixed worms for staining with a protein probe. The probe is supposed to bind specific cells within the pharynx (not around the pharynx) plus other cells in the body. However, the protein probe seems to accumulate only in the the buccal cavity and stains elsewhere in the worm’s body but the pharynx remains dark, unstained. The tissues around the pharynx also invariably tends to shrink. Given this, how could I make the protein probe to access the cells within the pharynx? Are there any better protocols that retain delicate structure of the pharynx, apart from the Modified F-R method? How could I stop shrinkage of the pharynx tissue?
I’ve tried using different formaldehyde and methanol concentrations, incubation times (2hr, 4hr, overnight) and temperatures (e.g. 4C, RT), number of washes, buffers (Buffer A, 1X PBS) but nothing seems to help. Perhaps chopping worms with razor would help? I would like to know if anyone has experienced similar issues and any tricks/advice for proper pharyngeal staining.
Thank you.

Since the pharynx is surrounded with a tight basal lamina layer, it is often more difficult to stain than other tissues.
This is especially true if you use a fixative like formaldehyde rather than precipitants like acetone or methanol.
If your label is working well on the cells outside of the pharynx, you might try a methanol-acetone fixation and see if the cells outside the pharynx still label. If you still do not see label in the pharynx, then the label may not label the cells you expect.
If you find you need to use formaldehyde, I recommend as little as possible (while still preserving label outside the pharynx). And do multiple cycles of fast freeze-thawing to help crack the cuticle, including the cuticle lining the inside of the pharynx.
I have tried staining dissected pharynxes once or twice - it was tedious and tricky and did not improve labeling in my hands.
Once you see the label in the pharynx, you can try and improve morphology - sequential alcohol rinses are often used mammalian tissues.
Good luck, Janet

Is it necessary to visualize in the pharynx at a specific stage, or just determine if it’s in the pharynx? If the latter, try freeze-cracking embryos. The basement membrane isn’t as tight then, for example in a 1.5fold embryo.


Thank you Dr Duerr and Dr von Stetina for your kind suggestions.

They need to be L4s/adults. I do at least 4 cycles of freeze-thawing. This is usually enough as rhodamine-phalloidin stain for actin shows nice staining of these fixed worms everywhere including the pharyngeal muscles. I tried chopping heads of fixed worms but this didn’t help much. Probably dissecting pharynx prior to fixation might help. I will also try out the acetone-methanol fixative.