Ab214428

The injured CAs underwent a series of processes, including excision, fixation, and embedding in paraffin. Then, the CAs were cut into 4 μm thick sections and analyzed through IF staining. Homoplastically, the treated HUVECs were fixed in 4% paraformaldehyde. Next, as previously described [20 (link)], the sections and HUVECs were incubated with primary antibodies against cathepsin B (1 : 100, ab214428, Abcam), cathepsin D (1 : 200, #2284 s, Cell Signaling Technology), ferritin (1 : 50, ab75973, Abcam), and TfR (1 : 50, ab269513, Abcam) at 4°C overnight. After washing three times on the following day, the samples were then incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (H+L) (1 : 300, A32790, Invitrogen, Carlsbad, CA, USA), Alexa Fluor 555-conjugated donkey anti-rabbit IgG (H+L) (1 : 300, A32794, Invitrogen), Alexa Fluor 488-conjugated goat anti-mouse IgG (1 : 300, A-11001, Invitrogen), and Alexa Fluor 555-conjugated goat anti-mouse (1 : 300, A-21424, Invitrogen) secondary antibodies at 37°C for 1 h. Next, 4,6-diamino-2-phenylindole (SouthernBiotech, Birmingham, AL, USA) was added to each section for coverslipping. Finally, the sections were observed under a fluorescence microscope (OLYMPUS BX50/BX-FLA/DP70; Olympus Co., Tokyo, Japan), and ImageJ software was used to quantify the fluorescence intensity.

The protein levels in cartilage tissues and cells were determined by referring to methods in previous literature [19 (link)]. Briefly, the protein lysates of brain tissues or cell samples were prepared with a radio-immunoprecipitation assay lysis buffer (P1003B, Beyotime, Shanghai, China) and 1% phenylmethylsulfonyl fluoride. Bicinchoninic acid (BCA) kits (Beyotime) were adopted for protein quantification. Subsequently, the proteins (15–50 μg) were separated with 4–20% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes with a pore size of 0.45 μm or 0.22 μm. Next, the membranes were blocked with 5% skim milk for 1 h and cultured with the primary antibodies gasdermin D (GSDMD)-N (dilution ratio of 1:1000, DF13758, Affinity Biosciences, USA), CTSB (dilution ratio of 1:1000, ab214428, Abcam), NLRP3 (dilution ratio of 1:1000, ab263899, Abcam), and GAPDH (dilution ratio of 1:10,000, ab181602, Abcam) at 4°C overnight. Afterward, the membranes were cultured with the secondary antibody anti-rabbit IgG (dilution ratio of 1:1000, ab205718, Abcam) for 1 h, and then visualized using an enhanced chemiluminescence reagent (#34,080, Thermo Fisher Scientific Inc., Waltham, MA, USA). The protein blotting was analyzed using the ImageQuant LAS 4000 (General Electric Company, Schenectady, NY, USA).