Alexa fluor 488 conjugated affinipure donkey anti rabbit igg h l

After isolation, HSGE cells were directly stained for characteristic markers and analyzed by flow cytometry. HSGE cells were harvested by centrifugation at 300g for 5 min and then washed two times in PBS. We fixed cells with 4% paraformaldehyde for 1 h at 4 °C and blocked for 2 h in 0.1% Triton X-100 and 5% BSA at room temperature. The following labeled antibodies were used: CK-8 (ab53280, Abcam, UK, 1:20) antibodies at 4 °C for 30 min, followed by Alexa Fluor 488-conjugated Affinipure Donkey Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, 1:200, USA) at 4 °C for 30 min.

HK2 cells were grown to 70% confluence on glass coverslips before immunofluorescence microscopy was performed. Cells were fixed for 8 min in 0.2% glutaraldehyde and 2% paraformaldehyde in PBS. This condition favors preservation of tubular endosomal structures although not to the extent seen by live microscopy. After fixation, cells were permeabilized in 0.05% (w:v) saponin (Sigma‐Aldrich, 558255), 0.5% (w:v) BSA and 50 mM NH₄Cl in PBS (blocking buffer) for 30 min at room temperature. The cells were incubated for 1 h with primary antibody in blocking buffer, washed three times in PBS, incubated for 1 h with the secondary antibody. Then, cells were washed three times in PBS, mounted with Mowiol (Sigma‐Aldrich, 475904‐M) on slides and analyzed by confocal microscopy.
We used anti‐LAMP1 (H4A3, USBiological, Life Sciences); anti‐EEA1 (Cell Signaling); anti‐Integrin β1 (TS2/16 Santa Cruz Biotechnology); anti‐Glut1 (ab15309 Abcam); anti‐c16orf62 (Thermo Fisher Scientific). As secondary antibody we used Cy3‐conjugated AffiniPure Donkey anti‐Mouse IgG H + L (Jackson Immuno Research); Cy3‐conjugated AffiniPure Donkey anti‐Rabbit IgG H + L (Jackson Immuno Research); Alexa Fluor®488‐conjugated AffiniPure Donkey anti‐Rabbit IgG H + L (Jackson Immuno Research).