Bin,
What neuronal marker are you using as the primary and what dilution are you using? Is the tissue perfused? Is it a frozen section or paraffin? It sounds like you have to dilute your primary and maybe your secondary more. You should try to titer each, the primary and secondary with the tissue you are using.
Jean Brennan
Research Specialist
Rothstein lab, Dept of Neurology
Johns Hopkins University
600 N. Wolfe St., Meyer 6-174
Baltimore, MD 21287
410-614-4119; FAX: 410-955-0672
[hidden email]>>> Bin Ma <
[hidden email]> 09/22/08 4:56 PM >>>
Hello everyone, does anyone have a good protocol for immunofluorescence staining of the primary neurons? I have problem in staining the mouse neurons. Please check the red channel in the attached picture. I use goat ant mouse cy3 to detect the antigen in the neurons.The problems are two: 1 is high red back ground. The 2 is many red spots everywhere on teh coverslips! in addition 1. it is good practice to centrifuge the primary and secondary antibody before application on cells? I tried , but sometims resulsr are not good. 2. the last step before mounting.The section should be washed briefly in water before mounting? should I let tehm air dry before mounting? I used gelatine. Thank you very much!
Bin Ma MD, PhD NYU school of Medicine |