Fitc conjugated anti mouse igg secondary antibody

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were used to analyze the expression of the target protein, and western blotting was developed using the gp85-specific mouse monoclonal antibody JE9 as the primary antibody. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Sigma, 1:5,000) was used as the secondary antibody.
For flow cytometry, L. plantarum cells were cultured in MRS broth overnight at 37°C. The cell pellets were sequentially incubated with gp85-specific mouse monoclonal antibody (1:800) and fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG secondary antibodies (1:5,000; Sigma). Finally, 3 × 10⁴ cells were analyzed with a FACS Calibur platform (Becton Dickinson, Oxnard, CA, USA) equipped with CellQuest software.

To measure the protein phosphotyrosine and acrosine availability, cell aliquots were fixed, permeabilized and blocked as described above (section actin polymerization). Subsequently, the samples were incubated for 1 h with specific anti-phosphotyrosine (4G10 Platinum, Sigma‒Aldrich) or anti-acrosine (ACR-2, Exbio, Praha, Czech Republic) primary antibodies diluted 1:100 in PBS with 1% BSA. After two washes in PBS, sperm cells were incubated with FITC-conjugated anti-mouse IgG secondary antibodies (Sigma‒Aldrich) diluted 1:100 in PBS with 1% BSA for another hour. Before the analyses, sperm cells were washed twice in PBS and diluted to a final concentration of 350 cells/µl. A blue 488 nm laser was used to excite the fluorescence of the labeled cells, and the emitted light was captured using a Green-B 525/30 nm filter.

Bacterial cultures were grown and induced as described above. Flow cytometry analysis was performed to verify surface display of AgE6 in L. plantarum. Cells harvested from approximately 500 μl culture were washed once with PBS. The pellet was then resuspended in a 1:250 dilution of the primary antibody anti-ESAT-6 (Abcam) in PBS/2% (w/v) BSA followed by incubation for 30 min at room temperature. Subsequently, the bacteria were washed two times with 600 μl PBS/2% BSA. After the second washing step, the pellet was resuspended in a 1:170 dilution of FITC-conjugated anti-mouse IgG secondary antibody (Sigma-Aldrich) in PBS/2% BSA followed by incubation for 30 min at room temperature, protected from light. The cells were then washed three times with PBS/2% BSA before subsequent analysis using a MACSQuant analyzer (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The data were analyzed using FlowJo software. Bacterial samples used for fluorescence microscopy were prepared in the same manner and analyzed with a Zeiss LSM 700 Confocal Microscope using Zen software. Image analysis was performed using the ImageJ plugin MicrobeJ (Ducret et al., 2016 (link)). The phase contrast images were used for determination of the shape and size of the bacterial cell, and the FITC signal intensities were extracted from the corresponding fluorescent images.