What is wrong with my fluorescent images(immunocytochemistry technical problem)?

02 February 2017 0 7K Report

i constantly see unfocused not sharp stainings.

i did immunofluorescence on polyester transwell inserts. an example of the image below is one that shows alpha-integrin staining(green).

the problem is when i try to focus and get clarity for a particular cell, the rest of the cells get out of focused. Is this bad antibody or do i need to use z-stack? would using less cells (confluency) solve the problem?

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