Alexa fluor 488 anti rabbit immunoglobulin g igg

For cultured cells, cells were washed with ice-cold PBS, fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS, stained using anti-Cas and anti-RelA as primary antibodies as well as Alexa Fluor 488 anti-rabbit immunoglobulin G (IgG) and Alexa Fluor 546 anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) as secondary antibodies, and then viewed with a confocal microscope (Nikon A1R system). 4′,6-Diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA) was used to stain the nucleus.
For osteocytes in mouse midshaft tibiae, mice were transcardially perfused with 4% paraformaldehyde in phosphate buffer (pH 7.4). Tibiae were immersed in the same fixative at 4°C overnight and decalcified in 10% EDTA (pH 7.4) at 4°C for 14 days. The samples were embedded in paraffin, sectioned with 5-μm thickness, and incubated with primary antibodies (anti-Cas, anti-RelA, and anti–acetylated RelA) at 4°C overnight. Alexa Fluor 488–conjugated goat anti-mouse IgG, Alexa Fluor 488 anti-rabbit IgG, and Alexa Fluor 594 anti-mouse IgG were used as secondary antibodies. Nuclei were counterstained using DAPI. Sections were mounted with Fluorescent Mounting Media (Dako, CA, USA). Quantitative 3D analysis of nuclear/total Cas was conducted using Imaris software (Bitplane, Zurich, Switzerland).

The quadriceps muscle was dissected out and post‐fixed in 4% PFA in phosphate‐buffered saline (PBS) for 1 h at 4°C. Samples were immersed in 30% sucrose/PBS overnight at 4°C and embedded in OCT compound (Sakura Finetek USA, Inc., Torrance, CA, USA). Frozen sections were cut with a cryostat (Leica Microsystems, Tokyo, Japan) at 10 μm and mounted onto MAS‐coated glass slides (Matsunami Glass, Osaka, Japan). Sections were washed with PBS and blocked with blocking solution (PBS containing 3% serum and 0.1% Triton X‐100) for 1 h; subsequently, sections were incubated overnight at 4°C in a mixture of primary antibody and rabbit anti‐laminin (Sigma‐Aldrich, 1:200). After washing three times with PBS, sections were reacted with secondary antibody (Alexa Fluor 488 anti‐rabbit immunoglobulin G [IgG], Invitrogen Corp., Tokyo, Japan, 1: 300) at room temperature for 1 h. After washing with PBS, sections were reacted with 4,6‐diamidino‐2‐phenylindole (DAPI) solution (Dojindo Laboratories, Kumamoto, Japan, 1: 2000) in PBS at room temperature for 30 min, washed three more times with PBS and then coverslipped with Vectashield (Vector Laboratories, Burlingame, CA, USA). Immunostained sections were observed under the fluorescence microscope FSX100 (Olympus Corporation, Tokyo, Japan) to measure the myofibre diameter of quadriceps muscles.