I am currently optimising a flow FISH protocol for an archaea species. The cells are fixed with and final concentration of 50% (v/v) ethanol before FISH hybridisation. The FISH protocol is carried out in cell suspension/pellet, and the resulted pellet is suspended using 0.5 mm needle in buffer solution prior to microscope check. There are several problems:
1) the cells tends to form aggregate;
2) the majority of archaeal cells are not labelled with the probes;
3) there is a very strong fluorescence background when checked under the microscope;
My assumption is that, the extracellular material of the archaeal cells is very stick, and this makes the cells get burst when physical force is applied. There might be other explanations. Can anyone offer some advice? Thanks in advice.
Yiyu
Hello! Were you able to find a solution? I'm experiencing similar problems.
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