Hello!
I’ve been optimizing antibodies for IF using the WTC-11 cells and have utilized both Matrigel and Vitronectin, but am still seeing non-specific binding in my secondary-only controls in both cell-containing and cell-free areas near the coverslip. I have read the online protocol about the potential for autofluorescence in Matrigel if prepared in advance, and have been following the protocol for seeding cells on Matrigel-coated glass, but wonder if you have any tips for reducing this non-specific binding within the cells in general.
Some details about my blocking:
Blocking for 1-2 hours at room temp
Blocking buffer is: 5% normal goat serum (NGS) in PBS with 2% BSA, and 0.5% Tween-20
Given the immunofluorescent staining you’ve published in Roberts et al., 2017 Figure S6 for Paxillin, for example, it looks like you were able to achieve clear signal-to-noise levels and minimal (if any!) non-specific binding. Any tips you can share to achieve this clean antibody staining are much appreciated!
Thank you for your help!