I'm trying to stain the chemoreceptors of male and female moths using Slifer's crystal violet method. I immersed the whole moths (which are about 1.5 cm long) in an aqueous solution of 10% formaldehyde for 24 - 48 hours. I washed them with distilled water and then I immersed them in an aqueous solution of 0.5% crystal violet for 1 - 24 hours and rinsed them twice with water. I cut and dried the antennae under a lamp for a few minutes, and transferred them to xylol. I mounted the antennae in Canada balsam.
After 2 hours, only the tips of the largest setae became stained in one antenna. But no other setae stain after 4, 10 and 24 h. The scales are bright purple, just like the dust on the surface of the antennae, but the chemoreceptors don't stain. It is obvious that in a male antenna most of the setae are chemoreceptors, so what could I be doing wrong?
Any ideas? Thank you.
Dear Ricardo,
As expert about fish gill chemoreceptor(Neuroepithelial ells,NECs),I would like to suggest the use of any marker to label these cells.Insect morphology is not my field of study.What is it the role of the setae chemoreceptors ? Are they nerve cell bodies .If yes, you can try a nerve marker by IHC.
Giacomo
Thank you for your answer, Dr. Zaccone.
In the case of insects, the olfactory neurons are housed in hairs (setae) on the surface of their antennae, palpi, etc. This method allows us to separate setae with mechanosensory functions (mecanoreceptors) from setae with chemoreceptive functions (chemoreceptors), since the latter have pores on the wall that allow the dye to enter. We can also tell apart contact chemoreceptors from olfactory chemoreceptors because there is only one pore at the tip of the sensilla in contact chemoreceptors, but several in olfactory ones.
But in this case, the method doesn't work for me.
Dear Ricardo again.
I think histology, but also TEM may help you to differentiate between the mechanosensitive and chemoreceptor cells.In fish I located in olfactory neurons Alpha G protein subunits by immunohistochemistry.But I do not know if he markers work for olfactory neurons of insects.
We planned to use TEM, but so far we haven't found anyone with experience working with insect setae. The Slifer method is cheaper, easier and faster, that's why we chose it. We may use those methods in the future, though.
Thank you again for your answer.
Dear Ricardo, I employed this technique many times for staining chemoreceptors on the antennae, legs, proboscis and other body regions in different insects. It only reveals sensilla having pores at their surface, but it does not stain cells. So, you do not need to fixate, neither the antennae, nor the insect. I used to cut the antennae (or the head) and put it between two gazes imbibed with the crystal violet solution, making attention that the dye does not reach the cut (otherwise it will diffuse inside). Then, I recovered the piece, rinsed it very quickly with distilled water and let it dry under a lamp on a filter paper. This is quite drastic, I know, and the antennae become twisted, but it works and porous sensilla are well stained. Please, do not hesitate in contacting me again for further details.
you can use cobalt chloride or commaci blue
https://ediss.uni-goettingen.de/bitstream/handle/11858/00-1735-0000-0006-AC70-5/gaaboub.pdf?sequence=1
Dr. Lazzari, thank you for the advise. I was following what was reported in Slifer 1960, but it is true, the dye only enters through the pores. I will try your method. Thank you.
Dr Abdallha, the method you used seems really interesting. Right now we don't have the time or the chemicals available, but we will try it in the future. Thank you.
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