Lsm 880 fcs confocal microscope

Stifle joints were fixed in 4% paraformaldehyde for 24 h, decalcified with 14% ethylenediaminetetraacetic acid (EDTA) for 14 d, and embedded in OCT compound (Sakura, Torrance, California).Sagittal sections of the stifle joint were prepared at 6 µm thickness with a Cryofilm type 3c (SECTION-LAB, Hiroshima, Japan). Inguinal fat pads from donor Pdgfra orPdgfrβ‐CreER^T2‐mT/mG mice were also dissected, embedded in OCT, and cryosectioned at 30 μm thickness. Routine safranin O / fast greenstaining of stifle joints section was performed with methods adopted from past work^{25 (link)–27 (link)}.
For immunofluorescent immunohistochemistry, sections were washed in 1× PBS, blocked in 5% normal goat serum (S-1000, Vector Labs, CA, USA) for 30 min, and incubated with primary antibodies specific for ACAN (1:100, ab3778, Abcam, MA, USA), COL10(1:100, ab58632, Abcam), CD31 (1:100, ab 28364, Abcam)and scleraxis (1:100, ab58655, Abcam) at 4 °C overnight. Sections were then incubated with Alexa Fluor® 647-conjugated secondary antibodies (1:200, ab150083, Abcam) or DyLight® 594 (1:100, DI-1594, Vector Labs), and mounted with mounting medium containing DAPI (H-1500, Vector Labs, Burlingame, CA)(Supplemental Table 1). Immunofluorescence images were acquired on a Leica DM 6B or LSM 880 FCS confocal microscope (Carl Zeiss, Oberkochen, Germany).