Fitc conjugated anti ha monoclonal antibody

Cells were allowed to adhere to the coverslips were fixed with cold methanol (−20°C), rehydrated with phosphate-buffered saline (PBS) and then blocked in 3% bovine serum albumin in PBS. Immunostaining was performed by incubating the fixed cells with the primary antibody for 1 h at room temperature. The following primary antibodies were used: fluorescein isothiocyanate (FITC)-conjugated anti-HA monoclonal antibody (1:400 dilution; Sigma-Aldrich), L8C4 (anti-PFR2 monoclonal antibody [MAb]; 1:50 dilution) (31 (link)), anti-TbSAS-6 polyclonal antibody (1:400 dilution) (11 (link)), anti-TbBLD10 polyclonal antibody (1:400 dilution), and YL 1/2 (1:1,000 dilution; Millipore). The following secondary antibodies were used: Cy3-conjugated anti-mouse IgG (1:400 dilution; Sigma-Aldrich), FITC-conjugated anti-rat IgG (1:400 dilution; Sigma-Aldrich), and Cy3-conjugated anti-rabbit IgG (1:400 dilution; Sigma-Aldrich). Cells were washed with PBS, mounted with Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole (Vector Labs), and imaged with an inverted fluorescence microscope (Olympus IX71) equipped with a cooled charge-coupled-device (CCD) camera (model Orca-ER; Hamamatsu) and a PlanApo N 60×, 1.42-numeric aperture differential inference contrast objective. Images were acquired using the Slidebook software (version 5; Intelligent Imaging Innovations).

Cells were washed once with PBS, settled onto glass coverslips for 30 min at room temperature, and fixed with cold methanol for 30 min at − 20 °C. Cells were rehydrated with PBS and incubated with the blocking buffer (3% BSA in PBS) for 1 h at room temperature. Cells were incubated with the FITC-conjugated anti-HA monoclonal antibody (1:400 dilution, Sigma Aldrich, Clone HA7) for 1 h at room temperature and washed three times with PBS. Coverslips were washed three times with PBS and mounted with DAPI-containing VectaShield mounting medium (Vector Labs). Immunostained cells were imaged with an inverted fluorescence microscope (Olympus IX71) equipped with a cooled CCD camera (model Orca-ER, Hamamatsu) and a PlanApo N 60 × 1.42-NA lens. Images were acquired using the Slidebook5 software and processed using the Adobe Photoshop software.