Alexa fluor 568 conjugated donkey anti mouse igg

Cells were grown to 70–80% confluence on cover glass slides (Menzel-Gläser; Braunschweig, Germany), washed twice with PBS and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 20 minutes at room temperature. The cells were permeabilized for 15 minutes with 0.4% Triton X-100 in PBS (PBST), and blocked with 6% bovine serum albumin (BSA) in PBST for at least 1 hour at room temperature. After removing of the blocking solution, the cells were incubated with mouse anti-human CD56 (NCAM) antibody (1:250 dilution; Cat # 318310 Biolegend) or mouse IgG1κ isotype control (1:250 dilution; Cat # 400122 Biolegend) at 4 °C overnight. The cells were washed three times with tris-buffered saline with 0.3% Tween 20 (TBST) and then incubated with the secondary antibody Alexa Fluor 568-conjugated donkey anti-mouse IgG (1:500 dilution; ThermoFisher Scientific) for 45 minutes at room temperature. The slides were washed three times with TBST, rinsed once with PBS and mounted in Vectashield mounting medium containing 1.5 μg/mL DAPI (Vector Laboratories, Burlingame, CA, USA). The slides were kept protected from light. There were examined and imaged under Zeiss fluorescent microscope and the images were processed using the Adobe Photoshop software.

Breast cancer tissues microarray slides were purchased from US Biomax (BC081120c). Each slide contained 110 specimens (100 cases of invasive ductal carcinoma and 10 normal breast tissue). The paraffin-embedded sections (5 μm thickness) were deparaffinized, rehydrated through descending grades of ethanol, and washed with PBS. For antigen retrieval, the slides were incubated in 10 mM Sodium citrate containing 0.05% Tween 20, pH 6.0 for 3 hours at room temperature and rinsed two times with 0.1 M PBS with 0.2% Triton X-100, pH 7.4 (PBST). To block non-specific protein binding, sections were incubated in 6% BSA in PBST overnight at 4 °C, then were incubated for 24 hours at 4 °C with mouse anti-human CD56 (NCAM) antibody (1:250 dilution; Cat # 318310, Biolegend) or with mouse anti-human NKp46 antibody (1:250 dilution; Cat # 331918, Biolegend). The primary antibodies were diluted in 2% BSA in PBST. After rinsing the sections with PBST for three times (15 min each), they were incubated for 2 hours at room temperature with Alexa Fluor 568-conjugated donkey anti-mouse IgG (1:500 dilution; Thermo Fisher Scientific, USA). After three rinses with PBST, Vectashield mounting medium containing 1.5 μg/mL DAPI was added to the slides and then covered with cover slips. The slides were kept away from light and the images were captured using a Zeiss fluorescent microscope.

Four-μm-thick histologic sections of the myocardium were deparaffinized in xylene, rehydrated, and subjected to 10 min heat-induced antigen retrieval in citric buffer (pH 6.0). Samples were blocked in 10% normal donkey serum (Jackson ImmunoResearch Inc., West Grove, PA) for 30 min at room temperature, incubated with 10 μg/ml of LpMab-12 overnight at 4°C, and then with Alexa Fluor 568-conjugated donkey anti-mouse IgG (Thermo Fisher Scientific Inc.) for 1 h at 37°C. Subsequently, the sections were incubated with goat anti-human LYVE-1 (10 μg/ml; R&D Systems, Inc., Minneapolis, MN) and mouse anti-α-sarcomeric actin (α-SA) (1:200 diluted; Sigma-Aldrich Corp.) for 2 h at 37°C, followed by fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat IgG and Alexa Fluor 647-conjugated donkey anti-mouse IgM (15 μg/ml each; Jackson ImmunoResearch Inc.) and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (1 μg/ml; Sigma-Aldrich Corp.) for 1 h at 37°C. The sections were then treated with 1% solution of Sudan Black B (Sigma-Aldrich Corp.) for 30 min at room temperature, and mounted in Vectashield medium (Vector Laboratories, Inc., Road Burlingame, CA). Images were acquired with Olympus FluoView FV100 laser scanning confocal microscope equipped with CCD camera (Bio-Rad Laboratories Inc.).