Alexa488 conjugated goat anti rabbit ig

Surgical sections of bone tissue were stained using a MEBSTAIN Apoptosis TUNEL Kit Direct (MEDICAL & BIOLOGICAL LABORATORIES CO., LTD., Nagoya, Japan), or stained with anti-single standard DNA (ssDNA) Rabbit IgG (#18731 1:50; IBL, Gunma, Japan) followed by Alexa488-conjugated goat anti-rabbit Ig’ (#A-11034 1:200; Invitrogen, Carlsbad, CA). Sections were also stained with Alexa Fluor 488-conjugated rat anti-mouse F4/80 (#B116640 1:100 BioLegend, San Diego, CA, USA) and goat anti-mouse TNFα (#K2911 1:100 Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), followed by Alexa488-conjugated donkey anti-rat Ig’ (#A-21208 1:200; Invitrogen, Carlsbad, CA) and Alexa568-conjugated donkey anti-goat Ig’ (#A-11057 1:200; Invitrogen, Carlsbad, CA). Sections were also stained with purified mouse anti-PCNA (#610664 1:100; BD Transduction Laboratories, Franklin Lakes, New Jersey) followed by Alexa488-conjugated goat anti-mouse Ig’ (#A-11029 1:200; Invitrogen, Carlsbad, CA). DAPI (#D1306 1:750; Wako Pure Chemicals Industries, Osaka, Japan) was used as a nuclear stain.

To detect changes in hair-bundle structure, cultures were stained with 0.5 μg/ml of rhodamine conjugated phalloidin in PBS containing 10% horse serum and 0.1% Triton X-100 (PBS/HS/T) for 1.5 hours at room temperature, rinsed three times in PBS, and mounted on glass slides with Vectashield mounting medium (Vector Laboratories, Peterborough, UK). Slides were viewed with a Zeiss LSM 510 confocal microscope. To label radixin or phosphorylated forms of the ezrin-radixin-moesin family of proteins, cultures were stained overnight at 4°C in rabbit anti-radixin (1:100 dilution) or rabbit anti-phospho-ERM (1:50 dilution), washed three times with PBS, labeled with a mixture of Texas Red phalloidin (Invitrogen, 1:500) and Alexa-488 conjugated goat antirabbit Ig (Invitrogen, 1:500) in PBS/HS/T for 2–3 hours at room temperature, washed, and mounted in Vectashield.

Serum C1q binding to HMGB1 was tested using HMGB1-coated microspheres. Briefly, HMGB1 protein (50 µg) was conjugated with biotin using the EZ-Link Sulfo-NHS-Biotin reagent (Thermo Fisher Scientific Inc.), and non-reacted biotin was removed using a PD-10 desalting column (GE Healthcare Life Sciences). Biotinylated human IgG was used as a positive control. Streptavidin-coated microspheres (Bangs Laboratories Inc.) were incubated with biotin-labeled HMGB1 for 1 h at RT. The HMGB1- or human IgG-coated microspheres were washed three times with GVB²⁺ buffer and incubated with 10% NHS for 30 min at 37°C. After washing, the microspheres were treated with mouse anti-HMGB1 Ab (R&D Systems) and rabbit anti-C1q Ab (Dako). Alexa 594-conjugated donkey anti-mouse Ig (Invitrogen) and Alexa 488-conjugated goat-anti-rabbit Ig (Invitrogen) were used as the secondary antibodies. A confocal microscope (FV1000, Olympus) was used for observing and capturing images of the fluorescent complexes. The bindings of C3c, a degradation product of C3b after activation, and of C5b-9 to HMGB1-coated microspheres were investigated using fluorescein isothiocyanate (FITC)-conjugated rabbit anti-C3c Ab (Abcam) and mouse anti-C5b-9 Ab (Quidel). mouse anti-HMGB1 Ab (R&D Systems) or rabbit anti-HMGB1 Ab (Abcam) was used for detecting HMGB1.