Anti mouse igg alexafluor 594

Immunocytofluorescence was performed to determine the localization of sAC in A. poculata sperm. Briefly, sperm were fixed in 4% (v/v) paraformaldehyde and incubated on a glass slide with either primary antibodies (0.55 µg ml⁻¹ anti-sAC and 2.5 µg ml⁻¹ anti-β-tubulin) diluted in blocking buffer or blocking buffer alone (controls). Next, sperm were incubated with secondary antibodies (anti-rabbit IgG AlexaFluor 488 and anti-mouse IgG AlexaFluor 594 at 4 µg ml⁻¹; Abcam), sealed under a glass coverslip with mountant containing DAPI, and imaged on a Leica DMi8 confocal microscope. Details are provided in the electronic supplementary material.

MeT-5A, HOMC-B1, DKO-MeT-5A, DKO-HOMC-B1, and Y-MESO-9 cells were cultured on glass coverslips and fixed using 4% paraformaldehyde solution for 20 min at room temperature. Cells were permeabilized with PBS containing 0.1% Triton X-100 and blocked using PBS containing 7% serum for 30 min. Then, cells were incubated with γ-catenin (desmoplakin; BD Transduction Laboratories, San Jose, CA, USA; 1:100 dilution) for 2 h at room temperature followed by fluorescence staining with anti-mouse IgG Alexa Fluor® 594 (Abcam, Cambridge, UK; 1:200 dilution) and Hoechst (Dojindo, Kumamoto, Japan; 1:200 dilution) for 1 h at room temperature. Images were acquired using BZ-II (Keyence) with a fluorescence microscope (BZ-X9000; Keyence).

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.