Anti rabbit a488

Femurs were fixed in 4% PFA (Electron Microscopy Sciences) for 4 h at 4°C, equilibrated in 30% sucrose/PBS. Bones were frozen and cryosectioned using Kawamoto's film method [55 (link)]. Six micrometer sections were stained with Abs in 0.1% Tween-20 (Sigma-Aldrich)/5% FCS/PBS after blocking with 5% FCS/PBS for 30 min. The following primary and secondary reagents were used: anti-CD8α (53-6.7, DRFZ), anti-CD3 (eBio500A2, eBioscience), anti-CD4 (GK1.5, DRFZ), anti-CD44 (IM7, DRFZ), anti-GFP (polyclonal rabbit, Life Technologies), digoxygenin-coupled anti-mouse/human fibronectin (polyclonal rabbit, Sigma-Aldrich, coupled in DRFZ), anti-rat-Alexa 555/Alexa 647 (polyclonal goat, Life Technologies), anti-rabbit A488 (polyclonal donkey, Life Technologies), anti-digoxygenin-Alexa 594 (DRFZ), streptavidin-Alexa 594 (Life Technologies). For the nuclear staining, sections were stained with 1 μg/mL DAPI in PBS. Sections were mounted with Fluorescent Mounting Medium (DAKO). All confocal microscopy was carried out using a Zeiss LSM710 with a 20×/0.8 numerical aperture objective lens and all images were generated by maximum intensity projection of 3–5 Z-stacks each with 1 μm thickness. Image acquisition was performed using Zen 2010 Version 6.0 and images were analyzed by Zen 2009 Light Edition software (Carl Zeiss MicroImaging).

Immunohistochemistry and quantification of images were performed as described in Adriaens et al. (2016) (link) using antibodies against γ-H2A.X (Cell Signaling 2577; 1/1400) and keratin 5 (rabbit polyclonal anti-keratin 5; Covance, PRB-160P-0100; 1/1000). For immunofluorescence the secondary antibody was anti-Rabbit-A488 (Life Technologies). Images were acquired with a ZEISS Axio Scan Z1 microscope using 20× and 40× objectives followed by stitching of the continuous fields using ZEN 2 software.