Alexa fluor 555 conjugated anti rabbit igg h l

A mouse monoclonal antibody against M protein and mouse polyclonal antibodies against NP protein and F protein were prepared in our laboratory. Anti-Flag, anti-β-actin, anti-α/β-tubulin, Alexa Fluor 488-conjugated anti-mouse IgG (H+L) and Alexa Fluor 555- conjugated anti-rabbit IgG (H+L) were purchased from Cell Signaling Technology (Beverly, MA, USA). The secondary antibodies against mouse or rabbit used for western blotting were purchased from Bioss Biotechnology (Beijing, China). 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Sigma-Aldrich (St. Louis, MO, USA). CCK-8 cell proliferation and cytotoxicity assay kit were purchased from Solarbio Life Sciences (Beijing, China). CytoD and nocodazole were purchased from Absin Biotechnology (Shanghai, China). Phalloidin-TRITC Conjugate was purchased from AAT Bioquest (Sunnyvale, CA, USA).

An Alexa Fluor 488-conjugated rabbit monoclonal antihuman insulin antibody (Cell Signaling Technology, Denver, United States) was used for flow cytometry. The primary antibodies utilized for immunocytochemistry and immunohistochemistry included mouse monoclonal antihuman insulin, rabbit monoclonal anti-human GCG, rabbit polyclonal anti-human c-peptide (Cell Signaling Technology), and rabbit polyclonal antihuman somatostatin (SST) (Novus Biologicals, Littleton, CO). The employed secondary antibodies were Alexa Fluor 488-conjugated anti-mouse IgG (H + L) and Alexa Fluor 555-conjugated anti-rabbit IgG (H + L) (Cell Signaling Technology).

Cell samples were harvested at indicated time points after infection and fixed with 4% paraformaldehyde. After infiltration with 0.25% Triton X-100 and blocking with 5% bovine serum albumin, the cells were incubated with indicated primary antibodies at 4℃ for 12 h and stained with Alexa Fluor 488-conjugated anti-mouse IgG (H+L) and/or Alexa Fluor 555- conjugated anti-rabbit IgG (H+L) (Cell Signaling Technology) at room temperature for 1 h. The nuclei were stained with DAPI (Sigma-Aldrich). The cells were washed five times with PBST (5 min/wash), then observed and photographed on a Nikon A1 fluorescence microscope (Nikon, Tokyo, Japan).