Mouse igg1 monoclonal antibody

Surface expression of TREM-1 was determined by flow cytometric analysis. THP-1 cells were co-incubated with the bacteria at an MOI of 10 and 100 for 4 and 24 h. After incubation, the cells were washed with phosphate-buffered saline. Fc receptors were blocked using human FcR blocking reagent (Miltenyi Biotec, San Diego, CA). Cells were stained using mouse anti-human TREM-1 antibody (Abcam, Cambridge, MA) followed by goat anti-mouse IgG Alexa Fluor 488 secondary antibody (Abcam). Mouse IgG1 monoclonal antibody (Abcam) was used as an isotype control. After washing, the cells were resuspended in phosphate-buffered saline with 0.5% bovine serum albumin. Cells were fixed in 2% formaldehyde in FACS buffer and analyzed using a BD Fortessa flow cytometer (Becton Dickinson). The results were analyzed using FlowJo (version 8.8.7).

For the current study, immunohistochemical staining was performed with the anti-c-CBL antibody, clone 3B12 (Lifespan Biosciences, Seattle, WA). The MACH4 Universal horseradish peroxidase-polymer detection system (Biocare Medical, Concord, CA) and 3,3-diaminobenzidine (DAB; Sigma-Aldrich, St. Louis, MO) were applied for protein visualization. All sections were counterstained with methylene blue (MB) (Sigma-Aldrich). Each staining series included an isotype-matched control using a mouse IgG₁ monoclonal antibody (Abcam, Cambridge, MA).