PLA was performed as described by the manufacturer (Sigma-Aldrich, St. Louis, MO). Rh30 cells (gift from Dr. Peter Houghton, Greehey Children's Cancer Research Institute, San Antonio, TX), and HeLa cells (ATCC, Manassas, VA) were interrogated with anti-GLI1 antibody (R&D Systems, Minneapolis, MN) and/or anti-H2A.Z antibody (Cell Signaling, Danvers, MA) for PLA.
PLA labeled cells were imaged on a confocal microscope (Zeiss 510 META, Carl Zeiss, Jena, Germany). Z-stacks were acquired to allow for three-dimensional rendering in Volocity software (Perkin Elmer, Waltham, MA). DAPI fluorescence images were processed in the EBImage package or the R statistical programming environment to better define the nuclear boundary. Differential Interference Contrast data from brightfield images were processed in Photoshop (Adobe, San Jose, CA) using high pass filtering and median filtering to allow for rough visualization of cell boundaries. No processing was done to PLA fluorescence. All images were handled equivalently in Volocity, they were rendered in “3D Opacity” mode and channel opacity, density and black levels were all set to the same between images. Each square in the grid is approximately 22 μm on a side.
PLA labeled cells were imaged on a confocal microscope (Zeiss 510 META, Carl Zeiss, Jena, Germany). Z-stacks were acquired to allow for three-dimensional rendering in Volocity software (Perkin Elmer, Waltham, MA). DAPI fluorescence images were processed in the EBImage package or the R statistical programming environment to better define the nuclear boundary. Differential Interference Contrast data from brightfield images were processed in Photoshop (Adobe, San Jose, CA) using high pass filtering and median filtering to allow for rough visualization of cell boundaries. No processing was done to PLA fluorescence. All images were handled equivalently in Volocity, they were rendered in “3D Opacity” mode and channel opacity, density and black levels were all set to the same between images. Each square in the grid is approximately 22 μm on a side.