# Tips for reducing non-specific antibody binding in WTC-11 cells on coated glass

**URL:** <https://forum.allencell.org/t/tips-for-reducing-non-specific-antibody-binding-in-wtc-11-cells-on-coated-glass/761>\
**Category:** hiPSC Culture Discussion\
**Created:** [April 23, 2025, 7:54pm UTC](https://forum.allencell.org/t/tips-for-reducing-non-specific-antibody-binding-in-wtc-11-cells-on-coated-glass/761 "2025-04-23T19:54:02Z")\
**Posts on this page:** 1\
**Page:** 1

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**Author:** ![MaggietheFuqua](https://avatars.discourse-cdn.com/v4/letter/m/87869e/32.png) [@MaggietheFuqua](https://forum.allencell.org/u/MaggietheFuqua)\
**Post date:** [April 23, 2025, 7:54pm UTC](https://forum.allencell.org/t/tips-for-reducing-non-specific-antibody-binding-in-wtc-11-cells-on-coated-glass/761/1 "2025-04-23T19:54:02Z")

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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!