Protein free tbs blocking buffer

HeLa cells expressing vector alone or Flag-RIEP were prepared for IF following a previously described protocol and cultured on glass coverslips coated with 1% gelatin (15 (link)). To label mitochondria, prior to IF, cells were labeled with 200 μM MitoTracker Red CMXRos for 30 min. Generally, cells were fixed with 4% paraformaldehyde at 4 °C for 20 min, then permeabilized with 0.1% TritonX-100 in the cold for 20 min. Cells were washed 3 times using TBST, then incubated with Protein-Free (TBS) blocking buffer (Thermo Scientific, 37570) at RT for 1 h. Then cells were incubated with primary antibody: anti-Flag, NPM1, endogenous RIEP-1, RIEP-216, or RPA194 (1:500 dilution) overnight in the cold room. After washing three times with TBST, cells were incubated with the appropriate secondary fluorescence-conjugated antibody (Alexa Fluor 488 Goat anti-Rabbit IgG or Alexa Fluor 568 Goat anti-Mouse IgG, 1:500 dilution, Thermo Fisher) diluted with blocking buffer for 1 h at room temperature. Cells were mounted on slides with mounting buffer containing DAPI (Thermo Fisher) for imaging using a Zeiss Zen confocal microscope 700 with 63× oil objective. Images were recorded with the same settings. Image quantification was performed using Image J.