Anti protein a polyclonal antibody

T. brucei cells were adhered to the glass coverslip for 30 min at room temperature, fixed with cold methanol (−20 °C) for 30 min, and rehydrated with PBS for 10 min. Cells were then blocked with 3% BSA in PBS for 30 min at room temperature and incubated with FITC-conjugated anti-HA monoclonal antibody (Clone HA-7, H7411, Sigma-Aldrich, 1:400 dilution) or anti-Protein A polyclonal antibody (Sigma-Aldrich, 1:400 dilution) at room temperature for 60 min. Cells on the coverslip were washed three times with PBS and then incubated with Cy3-conjugated anti-rabbit IgG (C2306, Sigma-Aldrich, 1:400 dilution) at room temperature for 60 min. Cells on the slides were washed three times with PBS and air dried. The slides were mounted in VectaShield mounting medium (Vector Laboratories) containing DAPI and examined using an inverted microscope (model IX71, Olympus) equipped with a cooled CCD camera (model Orca-ER, Hamamatsu) and a PlanApo N 60 × 1.42 NA lens. Images were acquired and processed using Slide-book software (Intelligent Imaging Innovations, Inc).

Cells were attached to the coverslips for 30 min at room temperature, fixed with cold methanol (−20°C) for 30 min, and rehydrated with PBS for 10 min. Cells on the coverslips were blocked with 3% BSA in PBS for 30 min at room temperature, and incubated with anti-CC2D polyclonal antibody (1 : 2000 dilution) [47 (link)], anti-CIF1 polyclonal antibody (1 : 1000 dilution) [22 (link)], anti-CIF2 polyclonal antibody (1 : 1000 dilution) [39 (link)], anti-Protein-A polyclonal antibody (Sigma-Aldrich, 1 : 400 dilution) or FITC-conjugated anti-HA antibody (Sigma-Aldrich, clone HA-7, 1 : 400 dilution) for 1 h at room temperature, or with anti-TbPLK polyclonal antibody (1 : 160 dilution) [17 (link)] for 1 h at 37°C. After the cells on the coverslips were washed three times with PBS, cells were incubated with Cy3-conjugated anti-rabbit IgG (Sigma-Aldrich, 1 : 400 dilution) for 1 h at room temperature. After three washes with PBS, the coverslips were mounted in DAPI-containing VectaShield mounting medium (Vector Laboratories) and examined using an inverted microscope (model IX71, Olympus) equipped with a cooled CCD camera (model Orca-ER, Hamamatsu). Images were acquired and processed with the Slidebook software (Intelligent Imaging Innovations).