Alexa fluor 488 conjugated donkey anti rabbit igg

4T1 cells were seeded in confocal cell culture dishes at a density of 1 × 10⁴ cells per dish. After culture in incubator for 12 h, HSN (final concentration: [pTBCB] = 50 µg mL⁻¹), HSN with DEVD (100 µM), HSN with DFO (100 µM), and PBS were, respectively, added to the cells. After incubation for 24 h, cells were gently washed three times with fresh PBS and then fixed with 4% paraformaldehyde. After fixation, cells were incubated with PBS containing 0.1% Triton X-100 (PBST) and then washed three times with ice-cold PBS. Afterwards, cells were incubated with 3% bovine serum albumin (BSA) in PBST for 30 min. Then cells were washed three times in PBS and incubated with primary cleaved caspase-3 antibody (Cell Signaling Technology) (1:1000 in PBST) in a humidified chamber at 4 °C overnight. After incubation, cells were washed three times in PBS and incubated with secondary antibody Alexa Fluor 488 conjugated donkey anti-rabbit IgG (Thermo Fisher Scientific) (1:1000 in PBST) at room temperature for 1 h. Then cells were washed with PBS and sequentially stained with BODIPY 665/676 (Thermo Fisher Scientific) (10 µM, 30 min) and DAPI. After mounted by Fluoromount aqueous mounting medium, cells were imaged under a LSM800 confocal microscope.

The antibodies used against the mouse target proteins were anti-PSGL-1 antibody (Q14242; Ray Biotech, USA), anti-EPX antibody (bs-3881R; Bioss Antibodis), Alexa Fluor 488-conjugated donkey anti-rabbit IgG (A21206), and Alexa Fluor 594-conjugated donkey anti-rat IgG (A21209) (ThermoFisher Scientific, Waltham, MA, USA). Peridinin chlorophyll protein complex (PerCP)-conjugated anti-Ly6G (127,654, Biolegend), allophycocyanin (APC)-conjugated CD11c (117,309, Biolegend), PE-conjugated anti-Siglec-F (552,126, BD Bioscience), FITC-conjugated anti-CD41 (133,904, Biolegend), and PE/Cy7-conjugated anti-CD62P (148,310, Biolegend) antibodies were used for flow cytometry and cell sorting. All drugs used for treatment were obtained from Sigma-Aldrich unless indicated otherwise.

Mice were killed by CO2 asphyxiation and eyes were enucleated. Eyes were fixed for 1 h in 4% PFA, then rinsed and sectioned at the limbus; the cornea and lens were discarded. Eyecups were incubated in 30% sucrose overnight at 4 °C, then embedded in OCT and sectioned (10 µm). Cryosections were blocked with PBS containing 1% horse serum, 0.1% Triton 1 h at room temperature and exposed overnight to rabbit anti-IAB1 antibody (019-19741, 1:200, Fujifilm Wako) at 4 °C. After washing, sections were incubated 2 h with an Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:500, ThermoFisher Scientific) and counterstained with Hoechst 33342 (1:1000, ThermoFisher Scientific). The slides were then washed, mounted, and viewed and photographed with a Leica DM550B fluorescence microscope (Leica Biosystems).

Expression vectors for FLAG-tagged wild-type and mutated forms of NRXN1α were transfected into HEK293T cells. Transfected cells were then incubated with Fc and NLGN1-Fc [59 (link)] (0.1 μM and 0.03 μM for Fig. 2 and Fig. 4, respectively) in DMEM containing 10% FCS, 2 mM CaCl2, and 1 mM MgCl2 for 30 min at room temperature. NLGN1 used in this study lacked splice segments ssA and ssB. After washing, cells were fixed with 4% PFA, immunostained with mouse anti-FLAG (1:1000, Sigma) and rabbit anti-human IgG (1:2000, Rockland, Gilbertsville, PA, USA) antibodies, and then visualized with Alexa Fluor 555-conjugated donkey anti-mouse IgG and Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibodies (1:400, Thermo Fisher Scientific). HEK293T cells were also transfected with an expression vector for FLAG-tagged NLGN1 and incubated with wild-type or mutated forms of NRXN1α-Fc (0.2 μM) (Fig. S2). After washing and fixing, cells were co-stained with antibodies against FLAG and Fc, followed by incubation with Alexa Fluor dye-conjugated secondary antibodies. HEK293T cell surface FLAG (Alexa Fluor 488) and cell surface-bound Fc (Alexa Fluor 555) signals were imaged using a confocal microscope and fluorescence densities of cells were quantified using the ImageJ 1.37 software. Statistical significance was evaluated by one-way ANOVA followed by post hoc Tukey’s test.

Primary antibodies used were; rabbit polyclonal anti-ß-gal antibody (1:2000, Thermo Fisher Scientific, A-11132), rabbit polyclonal anti-GFP antibody (1:1000, Thermo Fisher Scientific, A-11122) and goat polyclonal anti-Robo3 antibody (1:100–400, R&D systems, AF3076). Secondary antibodies used were; Alexa Fluor 488-conjugated donkey anti-goat IgG (1:200, Thermo Fisher Scientific, A-11055), Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:500, Thermo Fisher Scientific, A-21206), Alexa Fluor 647-conjugated donkey anti-goat IgG (1:500, Thermo Fisher Scientific, A-21447), Alexa Fluor 647-conjugated donkey anti-rabbit IgG (1:250, Thermo Fisher Scientific, A-31573) and Biotin-conjugated donkey anti-goat IgG (1:2000, Jackcon ImmunoResearch, 705–065–147).