Alexa fluor 488 conjugated anti mouse igg

The subcellular localization of the HBH-CFTR-3HA variant in CFBE Tet-on cells was examined as reported previously [74 (link)]. The cells, plated on a coverslip, were washed twice with a phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (FUJIFILM-Wako Chemicals, Japan) for 30 min at room temperature (RT). Cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min and blocked with 0.5% bovine serum albumin (BSA) in PBS for 20–30 min at RT. Cells were incubated for 2 h at RT with an anti-HA antibody (16B12, BioLegend, San Diego, CA, USA, 1:1000) in 0.5% BSA in PBS. Then, the cells were washed three times with PBS, and an Alexa Fluor^® 488–conjugated anti-mouse IgG (1:500, Jackson ImmunoResearch, West Grove, PA, USA) was added to the cells, which were then incubated for 1 hour at RT. After washing the cells three times with PBS, a DAPI solution (Peptide Institute, Inc., Japan) diluted in PBS (1:5000) was added to the cells, which were then incubated for 5 min at RT. After washing the cells three times with PBS, the cells were mounted with a Vectashield^® mounting medium (Vector Laboratories, Newark, CA, USA). The images were visualized and captured with an inverted laser confocal fluorescence microscope (SP8, Leica Microsystems GmbH, Germany).

5x10⁴ cells per well were seeded on Lab-Tek II glass chambers (Thermo Fischer Scientific). After 24 hours, cells were washed with ice-cold PBS, and they were fixed and permeabilized with ice-cold methanol (Sigma Aldrich) for 15 minutes at -20°C. Cells were incubated in PBS containing 2% BSA for 30 minutes at room temperature. Cells were incubated with the primary and the fluorescent antibodies for 1h at room temperature. The primary antibodies used were: rabbit polyclonal Syk (1:100) (sc-1077, Santa Cruz Biotechnology); mouse monoclonal anti-beta Tubulin (1:200) (T4026, Sigma). Primary antibodies were detected by Alexa Fluor 488-conjugated anti-mouse IgG (1:400) (715-546-151, Jackson ImmunoResearch laboratories) and APC-conjugated anti-rabbit IgG (1:200) (711-136-152, Jackson ImmunoResearch laboratories) antibodies, respectively. Nuclei were stained with Hoechst 33342 dye. After washing, the slides were mounted in VECTASHIELD Mounting medium (H-1400, Vector laboratories) and were analyzed visually using Zeiss Imager M2 microscope.

DyLight 800-conjugated anti-rabbit IgG (no. 5151S) and DyLight 800-conjugated anti-mouse IgG (no. 5257S) were purchased from Cell Signaling (Danvers, MA); Alexa Fluor 488-conjugated anti-rabbit IgG (no. 111-545-003) and Alexa Fluor 488-conjugated anti-mouse IgG (no. 115-545-003) were purchased from Jackson ImmunoResearch (West Grove, PA).

Cultured cortical neurons were fixed with 4% PFA for 1 h at room temperature and incubated with blocking solution containing 1% BSA and 0.1% Triton X-100 in PBS for 1 h. Primary antibodies, anti-TUJ1 (1:1000, 801201; BioLegend, San Diego, CA, USA) was applied overnight at 4°C. Fluorescent dye Alexa Fluor 488-conjugated anti-mouse IgG (715-545-151; Jackson Immunoresearch, West Grove, PA, USA) was used as the secondary antibody.

Cells were detached from tissue culture dish by incubation with 2 mM ethylenediaminetetraacetic acid (EDTA)/phosphate-buffered saline (PBS) and stained with first antibodies in 1% bovine serum albumin (BSA)/PBS for 30 min at 4 °C. As a negative control, cells were incubated with mouse IgG (MO75-3, MBL, Tokyo, Japan). After washing with 1% BSA/PBS, cells were incubated with secondary antibodies in 1% BSA/PBS for 30 min at 4 °C. After cells were washed with 1% BSA/PBS, stained cells were analyzed by using BD LSRFortessa X-20 (BD Biosciences) and FlowJ software (BD Biosciences). The primary antibodies used for flow cytometry were anti-DR4 antibody (Sigma-Aldrich, SAB4700541, DR-4-02, 4 µg/mL) and anti-DR5 antibody (Thermo Fisher Scientific, 14-9909-82, DJR2-2 (2-6), 2 µg/mL). The secondary antibody used for flow cytometry was Alexa Fluor 488-conjugated anti-mouse IgG (715-546-151, 1/250, Jackson Laboratories, Bar Harbor, ME, USA).

The processing, embedding, cryosectioning, and immunofluorescence staining of brain tissue were performed as previously described [34 (link)]. The final dilutions of primary antibodies—sheep anti-NPY (1:1000; Abcam, ab6173) and mouse anti-TH (1:1000; Immunostar, 22941)—were 1:1000. The following secondary antibodies were used (both at 1:500): Cy3-conjugated donkey anti-sheep IgG (1:500; Jackson ImmunoResearch, 713–165-147) and AlexaFluor 488–conjugated anti-mouse IgG (1:500; Jackson ImmunoResearch, 715-545-150). Sections were incubated for 5 min at room temperature with 1 μg/mL Hoechst 33342 (Invitrogen, H3570) in phosphate-buffered saline for nuclear staining, mounted on glass slides, and coverslipped with Vectashield Mounting Medium (Vector Laboratories, H-1000). From each mouse (at least 3 mice in total), 3–5 ARC or PVN sections were analyzed using LSM 780 or LSM 800 with maximal signal separation.

Extracellular vesicles-containing pellets were re-suspended in 1 mL of PBS containing 5μg/ml DiD(1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indodi- carbocyanine,4-chloro-benzenesulfonate salt Biotium, Hayward, CA) and incubated for 10 min. After ultracentrifugation at 100,000 g for 70 min at 4°C, the exosome-containing pellet was washed in PBS and centrifuged again for 90 min at 150,000 g to remove unincorporated DiD. To assess the kinetics of exosome internalization, labeled EV were re-suspended in DMEM supplemented with 10% vesicle-free FCS. DMEM-re-suspended vesicles were added to NTM cells (20 μg per 1.5x10⁷ cells) for various periods of time (0.5, 1, 2, 4, 8, 24 and 32 h). The cells were washed with PBS, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and stained with α-tubulin antibodies (62901 mouse polyclonal antibodies, Biolegend; 1:200 dilutions). Cells were then washed and Alexa Fluor 488-conjugated anti-mouse IgG (Jackson Immunoresearch Laboratories) were added as secondary antibodies for 1 h in the dark at room temperature. Samples were washed and mounted on slides in Fluromount-G mounting media (Southern Biotech). Images were analyzed with a FV1000-IX81 confocal microscope (Olympus, Tokyo, Japan) equipped with 60X objective. Pictured slides (30min / cell type) were analyzed for DiD labeled NPCE derived EV by Image J software