I should isolate single Plasmodium falciparum oocysts from mosquito midguts. In order to do that, I have to stain the midguts first. My present protocol is the following
1. a drop of 1% mercurochrome in PBS on a single midgut and incubation for 5 minutes
2. wash 3 times with PBS for 10 minutes each
3. mount on a slide and put a coverslip to flatten the sample for microscopy observation
For some reason, the oocysts are not stained as they should be (looking at pictures from other publications) and it's not possible to distinguish them by the rest of the sample. Everything has the same pink color.
Does anybody have some good detailed protocol to share? Also, my midgut samples (already dissected) are preserved in 70% ethanol. Could this affect the staining? Should I rehydrate them first or it doesn't matter?
We normally dissect the midguts in 1% NP-40 in PBS to permeabilise the tissue for 30 min followed by the staining with mercurochrome for at least another 30 minutes. Also we only use 0.1% mercurochrome for staining. Hope that helps although my answer comes quite late.
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