Immunofluorescence 

parafilm

If you do Blocking properly you will get good result with nonspecific background. so better do blocking and you will get the clean result and you do not have to worry about unspecific binding that may give you false positive result. hope you will get it. Thanks

To make epitope more visibility

parafilm

@@yfn9408 m confused, if parafilm is placed on top of the cells ...then how primary antibody will interact with the cells crossing parafilm??

Catalina Rubio PFA is used to fix the cells and its inner structures so you can target with antibodies and be able to see them under the lens

The culture media most probably

do you have the answer already now bro? I’m also having the same question :(

same question

It's best to leave it wet. maybe if there's a lot of water (maybe, a drop?) on the opposite side of the cell side, you can tap that side lightly to a kimwipes so the droplet won't affect your mounting. If we were to leave it too dry, the cells can be destroyed, sheared or shrunk. hope it helps. @@dr.tranngocthien

Probably because this is a mounting procedure for immunostaining eukaryotic cells. Prokaryotes don't have any organelles or other large structures inside the cell to fluorescently label and look at under a microscope so there is no need to go through with a permealization procedure like this. Even if you wanted to label something in the cell membrane/wall, bacteria are small enough that it would be very hard to distinguish puncta with the resolution of traditional microscopes, so you wouldn't really get much useful information by immunostaining. In a microbiology setting, it is typically much more useful, cheaper, and easier to place cells directly from culture to a slide, fix them to the slide with a bunsen burner, and use common stains (carbol fuschion, crystal violet, malachite green) to see bacteria and any potential structures.

A first antibody is needed in order to recognize the protein you want to observe. Then, you use a specific second antibody which is gonna recognize the first antibody. In the second antibody, there is a fluorochrome ( it is fluorescent in the microscope ). I think i didn't say something wrong, but i'm not sure, also sorry if my english is quite bad :)