Anti rabbit igg alexafluor 647

Protein expression and distribution in hPDLSCs and periodontal tissues were investigated using immunofluorescence. Cell samples were rinsed in PBS and fixed with 4% PFA after 2 h of DAP treatment. Next, permeabilized the membrane of cells with 0.5% Triton X‐100 and blocked the samples in PBS containing 1% BSA for 1 h. Following incubation overnight at 4°C with the primary antibody Phospho‐p38 (ZEN BIO), cells were incubated with the fluorescein‐conjugated anti‐rabbit IgG AlexaFluor®647 (Abcam) for 2 h. The cytoskeleton was stained with phalloidin (Thermo Fisher Scientific) and the nuclei was stained with 4′, 6‐diamidino‐2‐phenylindole (DAPI; Sigma). The preparation of tissue slices was identical to that used for immunohistochemistry. Rabbit anti‐CARD4 antibodies (Bioss Antibodies) were used as primary antibodies. Anti‐rabbit IgG (Alexa Fluor® 647, Abcam) was applied as secondary antibodies. Nuclear staining was performed with DAPI (Sigma). The fluorescence staining of cells and tissues was examined using CLSM (FV3000, Olympus, Japan).

OVCAR8 cells were incubated for 30 min at 37 °C in medium containing 1 µM of 1H11 or IgG. Cells were washed with ice-cold HBSS, incubated with Wheat Germ Agglutinin (WGA) conjugated with Alexa Fluor (Thermo Fisher Scientific, Waltham, MA, USA), then washed before and after fixing with 4% PFA/PBS. Cells were blocked with 5% (w/v) BSA and 0.05% (w/v) saponin, then incubated overnight with primary antibodies diluted in blocking solution, anti-LAMP1 antibody (Abcam, Cambridge, MA, USA), or anti-EEA1 (Abcam, Cambridge, MA, USA). Cells were then washed before and after incubation with secondary antibodies (anti-mouse IgG (Alexa Fluor 594) (Abcam, Cambridge, MA, USA) or anti-rabbit IgG (Alexa Fluor 647) (Abcam, Cambridge, MA, USA). Finally, the slides were mounted using Prolong^® gold antifade mountant with DAPI (Thermo Fisher Scientific, Waltham, MA, USA). Images were acquired using the Zeiss LSM 880 Confocal Microscope (ZIESS, Waltham, MA, USA) at the Advanced Microscopy Facility at the University of Virginia.