Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo

confocal analysis


  • Please log in to reply
2 replies to this topic

#1 beny

beny

    member

  • Active Members
  • Pip
  • 19 posts
2
Neutral

Posted 06 July 2011 - 05:05 AM

Hi
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.

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,831 posts
415
Excellent

Posted 06 July 2011 - 07:38 PM

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.

#3 chabraha

chabraha

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 137 posts
2
Neutral

Posted 11 July 2011 - 09:39 PM

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




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.