Goat anti mouse igg antibody

The level of the osteocalcin (OCN) expression on the materials’ surfaces was assessed at 4 weeks post-seeding by immunofluorescence microscopy. Thus, after fixing, permeabilizing and blocking the cells, as mentioned in Section 2.4, the cell-populated samples were incubated for 2 h with primary anti-osteocalcin antibody (Santa Cruz, 1:50 dilution), washed with PBS and, subsequently, incubated in the dark with goat anti-mouse IgG antibody coupled with AlexaFluor 488 (1:200, Invitrogen, Eugene, OR, USA), for 30 minutes. Finally, the nuclei were labeled with DAPI (Sigma-Aldrich Co., Steinheim, Germany) for 10 mins. The labeled samples were visualized under the fluorescence inverted microscope (Olympus IX71, Olympus, Tokyo, Japan). Representative microscopic fields were captured using the Cell F (Version 5.0, Olympus Soft Imaging Solutions, Münster, Germany) acquisition system and the mean fluorescence intensity was measured using ImageJ software (Version 1.52d, National Institutes of Health, Bethesda, MD, USA).

Lymphocytes from the lungs were isolated and stained as described previously (32 (link), 41 (link)). For intracellular staining, cells were stimulated with heat-killed bacteria (65℃ for 30 min) at the indicated multiplicity of infection for 16 h at 37°C with Golgi Plug/Stop added for the last 5 h, and then stained as described (32 (link), 41 (link)). Cells were stimulated ex-vivo with PMA (50  ng/mL) and ionomycin (1 µg/mL) for 5 h at 37°C in the presence of BD GolgiStop to detect the total T cell responses. Enzyme-linked immunosorbent assay (ELISA) kits were used for detection of IL-17A and IFN-γ from BALF following the manufacturer’s protocol (BioLegend).
The bacteria-specific antibody response was measured by ELISA as previously described (41 (link)). Briefly, 96-well ELISA plates were coated with heat-killed bacteria. Wells were blocked with 2% nonfat milk in PBS and serial dilutions of BALF were applied to the wells. Plates were incubated with sera for 1 h at 37°C. Antibodies were detected with goat antimouse IgG-HRP (H+L). For detection, the TMB peroxidase substrate reagent set was used according to the manufacturer’s instructions (BioLegend). Optical densities were read at 450 nm with a microplate reader (Molecular Devices SpectraMax 190 Microplate Reader). Bound antibody in BALF was detected with goat antimouse IgG antibody (Invitrogen).

After treatment, cells were fixed with 4% paraformaldehyde for 30 min at room temperature. Cells were then permeabilized with 0.2% Triton X-100 for 15 min. After three washes, the permeabilized cells were blocked with 5% bovine serum albumin (BSA) for 30 min. Then, cells were incubated with 1:100 dilution of Stat3 (mouse antibody) overnight at 4°C. The next day, cells were incubated in a 1:100 dilution of goat anti-mouse Ig G antibody (Invitrogen) for 1 h at room temperature. The nuclei were counterstained using DAPI (Beyotime, China). Images of cells were visualized under fluorescence microscope (Olympus, Japan).

Mosquitoes were anaesthetized on ice and then were inject with 250 nanoliters of 20% paraformaldehyde for hemocyte fixation in midgut basal lamina. After 20 minutes, midguts were dissected in 4% paraformaldehyde diluted in phosphate-buffered saline (PBS) (13 mM NaCl, 0.7 mM Na2HPO4, 1 mM NaH2PO4 at pH 7.2) (PBS). The remaining midguts were fixed in the same solution for 20 minutes, and then washed three times in PBS and then incubated with blocking solution PBSBT (1× PBS + 1% BSA + 0.1% Triton X-100) for 15 minutes at room temperature. Samples were then incubated overnight with 4G2 monoclonal antibody for Flavivirus E protein (ATCC: HB-112, used at 1:50 in PBST) at 4°C. Midguts were washed three times with PBSBT (5 min each) and incubated for 2 h with constant rocking at 25°C with goat anti-mouse IgG antibody (Invitrogen). Midguts were washed three times with PBST (5 min each) and incubated for 15 min with DAPI (Molecular Probes, 1:500), and phalloidin-rhodamine (Molecular Probes, 1:500). Then the midguts were washed in PBS and placed onto slides. Images were obtained with an LSM 880 microscope (Zeiss).

Jurkat P116 or Jurkat P116 ZAP70-GFP cells were stimulated with 0.5 μg/ml CCL19 or CCL21 in presence of 0.9 μg/ml mAb24 [anti-CD11a+CD18 antibody [24] (ab13219) (abcam)] for 10 min at 37°C in HBSS. Subsequently, cells were put on ice for 30 min, fixed with 4% PFA and stained with secondary goat-anti-mouse-IgG antibody coupled to Alexa647 (Life Technologies) in HBSS and 3% BSA. Cells were washed in HBSS and fluorescence was analyzed on a LSRII flow cytometer (BD Biosciences). Quantification was done using FlowJo7 software.