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confocal analysis - image analysis (Jul/06/2011 )

i am new in doing confocal and wants to ask some question regarding the same.
i am using virus to infect my cell and then score for viral protein by specific antibody.
i take same no of cells for infected and control sample.
fix them followed by Permeabilization, primary and secondary antibody treatment with washing after each step and then make the slides.
when i start looking in fluorescence microscope normally i see same type of staining in both.
then i do confocal with keeping power at low voltage such that control show minimum background and then score for my sample with same parameters.
my boss always ask how sure are you about the infection.whether what u see is background or protein is really expressing.
i have a concern as i see same type of staining if i use normal florescence microscope which does not have any shutter or filter option to change any parameters.
can i take the confocal result as positive or not by keeping minimum in control and scoring for test samples.


You need two things in your experiment and they are both controls:

You need proper antibody controls - these include "no primary" and "no secondary" controls done on both infected and uninfected cells. If you do not see any signal with any of these controls you can assume that the staining you are seeing in your infected and stained cells is from the antibody binding to your viral protein.


just to add..........several DNA viruses encode their own Fc include an isotype control.......especially if your using a mouse monoclonal, just to make sure you are detecting your viral protein of interest.