Immunofluorescence Assay for Endosomal Markers

Fibroblasts were cultured in chamber slides for 72 hrs after passage and washed with PBS twice, fixed with 4% PFA at room temperature for 15 min, and permeabilized using 0.1% saponin. Slides were blocked with 5% donkey serum and 1% BSA for 30 min, incubated in primary Abs overnight at 4°C followed by incubation in appropriate secondary Abs, mounted in Vectashield solution containing DAPI (Vector Laboratories). Antibodies used for this method were: rabbit polyclonal anti-EEA1 Ab (Abcam), rabbit polyclonal anti-Rab11 Ab (Abcam), rabbit monoclonal anti-Rab7 AB (Cell Signaling), mouse monoclonal anti-CD63/LAMP3 Ab (H5C6; Developmental Studies, Hybridoma Bank), and Alexa fluor secondary donkey anti-rabbit/mouse Abs (Invitrogen). Cells were imaged with a Zeiss 510 META confocal laser-scanning microscope. For quantification of CD63 staining, 6000 cells/well were seeded in 96-well black/clear bottom plates and stained with anti-CD63/LAMP3 Ab, followed by donkey anti-mouse Alexa fluor secondary antibody. Cells were then imaged on the InCell 2200 Analyzer and quantified as described elsewhere [11 (link)].