Fitc conjugated goat anti mouse igg secondary antibody

Expression levels of ICAM-1 (on the EC surface), MUC1 and CD43 (on the cancer cell surface) were analyzed by flow cytometry (Accuri C6 flow cytometer, BD Bio-sciences). Quantification was made by measuring the geometric mean fluorescence. For immunofluorescence staining, glass coverslips were coated with 25 µg/ml human fibronectin. Cells were fixed with 2% paraformaldehyde, and processed for indirect immunofluorescence microscopy.
For measuring expression levels of ICAM-1 and MUC1, ECs or cancer cells were incubated with the primary antibody and then with FITC-conjugated (goat anti-mouse IgG) secondary antibody (Jackson ImmunoResearch, USA). The primary antibodies are a Human monoclonal antibody to ICAM-1 [26] (link) or an anti-MUC1 monoclonal antibody C595 (Santa Cruz Biotechnology, Santa Cruz, USA). The anti-MUC1 antibody recognizes a tetrapeptide motif within the protein core of the MUC1 molecule.
For CD43 expression level, cancer cells were incubated with a monoclonal antibody CD43-clone L10 labeled with FITC (Invitrogen, USA), which reacts with the extracellular domain.

Cultured cells were stained directly on culture plates. Cells were washed with cold PBS, fixed with 4% paraformaldehyde for 20 min, permeabilized and blocked in 0.1% Triton X‐100, 3% bovine serum albumin (BSA), incubated at room temperature for 1 hr with primary antibody for myosin heavy chain (MHC) (MF‐20; Developmental Studies Hybridoma Bank), washed with PBS, incubated at room temperature with FITC‐conjugated goat anti‐mouse IgG secondary antibody (#115‐095‐003, Jackson Immunoresearch), washed with PBS, and mounted in mounting medium with DAPI (Vectashield, H‐1200; Vector).
Myotube size and fusion were quantified in wells double stained for MHC (MF20) and nuclei (DAPI) by morphological analysis on five representative images of each sample obtained using a Keyence BZ‐X710 All‐in‐One Fluorescence Microscope with a 20× objective. Images were analyzed using ImageJ (NIH). Measurements were taken for total nuclei count, myotube count (# of MHC+ cells with 2+ nuclei), myotube nuclei count (# of nuclei in MHC+ cells that contain 2+ nuclei), and diameter. Diameter measurements were taken by averaging three separate measurements taken along the entire length of the myotube. Fusion index was defined as the ratio of myotube nuclei to total nuclei per field.