Alexa fluor 594 conjugated goat anti mouse igg antibody

HT-29 and Caco-2 cells were seeded onto 24-well plates. After treatment, cells were fixed in 4% paraformaldehyde for 15 min, washed with PBS, and permeabilized with or without 0.1% Triton X-100 (Beyotime) for 30 min. After blocking in 10% goat serum for 60 min, slides were incubated with a rabbit ABCG2 antibody (1:40) or a PDZK1 antibody (1:100) overnight at 4 °C. Samples were then incubated with Alexa Fluor 594-conjugated goat anti-mouse IgG antibody (Invitrogen) for 2 h, and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Samples were observed under a fluorescence microscope (Leica, Solms, Germany).

Human-induced pluripotent stem (iPS) cell lines (201B7 and 606A1) were purchased from Riken (Ibaraki, Japan). The human chondrocyte cell line (C28/I2) was purchased from Merck (Darmstadt, Germany). R-17F was provided by Dr. Kawasaki, Ritsumeikan University (Shiga, Japan). 4’,6-diamidino-2-phenylindole (DAPI) and Propidium iodide (PI) was purchased from DOJINDO (Kumamoto, Japan). Alexa Fluor 594-conjugated goat anti-mouse IgG antibody was purchased from Invitrogen (Tokyo, Japan). BlotGlyco beads were from Sumitomo Bakelite Company Ltd. (Tokyo, Japan). Other solvents and reagents were of the highest grade commercially available.

rPLY-treated neutrophils were fixed and permeabilized using a cell fixation and permeabilization kit (Thermo Fisher Scientific) according to manufacturer instructions, followed by incubation of the cells in a blocking solution (Thermo Fisher Scientific) for 30 min. For NE detection, samples were stained with rabbit anti-NE antibody (Abcam) in the blocking solution. After overnight incubation at 4 °C, the secondary AlexaFluor 488-conjugated goat anti-rabbit IgG antibody (Thermo Fisher Scientific) in blocking buffer was added, followed by a 2-h incubation in the dark. For detection of the P2X₇ receptor and PLY, samples were stained with goat anti-P2X₇ receptor (Abcam) and mouse anti-PLY antibodies (Abcam). After overnight incubation at 4 °C, the secondary AlexaFluor 488-conjugated bovine anti-goat IgG antibody (Jackson ImmunoResearch, West Grove, PA, USA) was added, followed by incubation for 2 h. Samples were then washed with PBS and incubated with AlexaFluor 594-conjugated goat anti-mouse IgG antibody (Thermo Fisher Scientific) and observed with a confocal laser-scanning microscope (Carl Zeiss).

Cells were fixed with 4% paraformaldehyde (4% PFA) for 30 min at room temperature (RT), permeabilized with 0.1 percent Triton X-100 for 15 min at RT, washed with PBS and blocked for 1 h with heat-inactivated 5% horse serum in PBS (PBS-HS). Cells were then incubated overnight at 4 °C with mouse monoclonal antibodies to IAV nucleoprotein (clone HB65, American Type Culture Collection, Manassas, VA, USA) diluted in PBS-HS at 0.2 µg/mL. Cells were washed and incubated for 1 h at room temperature with AlexaFluor-594-conjugated goat anti-mouse IgG antibody (0.2 µg/mL) and NucBlue Live ReadyProbes Reagent (ThermoFisher Scientific, Asheville, NC, USA). Images were collected using a Leica DMi8 fluorescent microscope (Leica, Wetzlar, Germany), and quantitative analysis was conducted using the ImageJ (NIH, Bethesda, MD, USA) Threshold, Watershed and Particle Analyzer tools, adapted from [28 (link)] (n = 4). The total number of cells was determined by the nucleus count per 0.6 mm². The number of infected cells per 0.6 mm² was determined using NP staining. The infected-to-total cell ratio was used to calculate relative infectivity. Relative viability was defined as the ratio of the number of treated infected to treated uninfected cells for each SMKI. Prism 9 Heatmap (GraphPad, San Diego, CA, USA) function was used for visualization.

To evaluate the phenotypes of the MSCs encapsulated within the Osteogel, an immunostaining of CD90 was performed. Cells were cultured in the Osteogel for 7 days, washed with DPBS, then fixed with 4% PFA and blocked with 10% Normal Goat Serum for 2 h. Primary anti-human CD90 antibody (Clone 5E10; Sigma-Aldrich GmbH, Schnelldorf, Germany; 1:100 dilution) was added to the cells overnight at 4 °C. Washed using PBS containing 0.5% Tween 20, Osteogels were incubated with secondary AlexaFluor^® 594-conjugated goat anti-mouse IgG antibody (ThermoFisher Inc., Waltham, MA, USA; 1.0 mg/mL, 1:200 dilution) and 4′,6-diamidino-2-phenylindole DAPI (ThermoFisher Inc., Waltham, MA, USA; 1:400 dilution). After intensive washing, the immunofluorescent staining was documented by Keyence BZ-9000 Fluorescence Microscope (Keyence GmbH, Neu-Isenburg, Germany).