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To confirm that the chondrocytes subjected to CTS were not dedifferentiated, immunohistochemistry was performed with type II collagen and type I collagen staining. Cells were fixed with 4% paraformaldehyde for 15 min at 48 h post-CTS. The cells were incubated with primary antibodies against type II collagen and type I collagen for 1 h at 37°C (SC-25974, SC-7764, Santa Cruz, CA, USA). Then the cells were incubated with a Texas Red-conjugated secondary antibody (SC-2783, Santa Cruz, CA, USA) for 30 min. Cellular nucleus were stained with Hoechst 33242 (Pierce, Rockford, IL, USA). The image was captured by using a Nikon E800 fluorescence microscope.

Double fluorescent staining was performed to examine caspase-3 activity in microglial cells following cerebral I/R as described previously 9 (link),13 (link). Briefly, brain tissues were immersion-fixed in 4% buffered paraformaldehyde, embedded in paraffin, cut at 7 μm, and stained with a specific anti-cleaved caspase-3 antibody which was labelled with FITC. After washing, the sections were incubated with anti-ionized calcium-binding adapter molecule 1 (IBA1; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 25°C for 1 hr to stain activated microglial cells. After washing, the sections were incubated with Texas Red conjugated anti-goat antibodies (sc-2783; Santa Cruz, Santa Cruz, CA, USA) for 1 hr at 25°C. The sections were then incubated with DAPI for staining the nucleus. The sections were covered with fluorescence mounting medium (Vector Labs, Burlingame, CA, USA). The images were viewed on an EVOS-fI digital inverted fluorescent microcopy (Advanced Microscopy Group, Bothell, WA, USA). Fields of cortex were randomly examined using a defined rectangular field area for analysis of microglia activation.