Anti mouse igg r antibody

With informed consent, human lobar pulmonary arteries were harvested from a patient who underwent a pulmonary lobectomy and immediately frozen at −80 °C. All procedures performed on human experiments were approved by the Research Ethics Committees of Ewha Womans University, and experiments were conducted in accordance with the approved guidelines. Frozen sections were incubated with 3 × 10¹¹ pfu of SPs-displaying phages for 3 h at 37 °C and then washed to remove unbound phage. After fixation, frozen sections were blocked and incubated with an anti-M13 bacteriophage antibody (1:200, Abcam, USA) overnight at 4 °C. Anti-mouse IgG-R antibody (1:500, Santa Cruz Biotechnology, USA) was used as a secondary antibody. DAPI was used for nuclear counterstaining. After mounting, tissue slides were visualized by fluorescence microscopy.

Three days after carotid artery ligation surgery, 2 × 10¹¹ pfu of SPs-displaying phages were injected intravenously and allowed to circulate for 10 min. The carotid arteries and aorta were collected for en face staining. Isolated carotid arteries and aorta were fixed. After blocking with 10% donkey animal serum in PBS, tissues were incubated with an anti-M13 bacteriophage antibody (1:200, Abcam, USA) overnight at 4 °C. The tissues were then incubated with a secondary anti-mouse IgG-R antibody (1:500, Santa Cruz Biotechnology, USA) for 2 h at room temperature. Phages attached on the endothelium were detected by LSM Pascal confocal microscope (Carl Zeiss, Germany). For Z-stack imaging of phage localization on endothelium, tissues were observed on Leica TCS SP8 confocal laser scanning microscope (Leica, Germany). All images were analyzed with Leica software (LAS X) and final images were obtained by using Huygens software.

For in vitro binding studies of SPs-displaying phages, iMAECs and HUVECs were exposed to LSS or OSS for 48 h. The cells were biopanned individually for 1 h with 6 × 10¹⁰ pfu of each SPs-displaying phage at 37 °C. Cells were fixed and blocked with 10% donkey animal serum in PBS. For detection of phages bound to surface of ECs, immunofluorescence staining was performed without permeabilization. Cells were incubated with an anti-M13 bacteriophage antibody (1:200, Abcam, USA) overnight at 4 °C. Anti-mouse IgG-R antibody (1:500, Santa Cruz Biotechnology, USA) was used as a secondary antibody. Phages bound to ECs were detected by Olympus BX51 (Olympus America). All images were analyzed with Motic Images Plus software (MIPlus 2.0) and the percent of phage positive ECs was quantified.