Is there a way to clear stain the cytoskeleton for observation in TEM? I usually do en bloc staining(rOTO) using Drosophila. This method stains the membrane clearly but does not allow me to see the cytoskeleton. I would like to know if there is a method that balances the two or stains the cytoskeleton more clearly.
You can stain the ultrathin sections with Lead citrate according to Reynolds and with Uranyl acetate (this is a classic technique that has a lot of variations and you have to choose the most appropriate for your samples). In our case, it works well adding the contrast to the sections and making the citosceleton structures visible quite well.
Serge Voychuk Thanks for the answer. It sounds like a better way to stain the ultrathin sections than the block.
In fact OTO (osmium tetroxide) was never used for staining and it sounds a bit confusing to “do en bloc staining with rOTO” or something like that. The main goal of OTO is fixation of the lipids of the tissues. And it used mainly as a second step of tissue/cells culture fixation procedure after fixation with glutaric aldehyde or else. Thus you have to use OTO any way to fix your samples, but don’t hope to get a good contrast after this procedure. Moreover the good contrast that one may really have after OTO fixation is an artefact, because it means that you poorly washed your samples after OTO fixation and OTO crystals are attached to membranes. It’s not good.
Albeit the sample block become black after OTO fixation, the ultrathin sections, prepared from it and not stained/contrasted with Uranyl acetate (UA) and Lead citrate (LC), should be almost transparent. I never saw the protocol where OTO was used as a stain of the ultrathin sections. Therefore, I suggest you not to use term “staining with OTO” instead use “fixation with OTO and contrasted with UA and LC”.
- Badges
- Science topic
- Similar topics
- Engineering
- Chemical Engineering
- Membranes