Can get signal immunostaining solution

Slides were pretreated with antigen-retrieval reagent (Immunoactive; Matsunami Glass Ind, Osaka, Japan) at 60°C for 16 h, followed by blocking serum for 30 min. Then, sections were immunostained with anti-P16^INK4a antibody (abcam, ab54210, 0.1 μg/ml), anti-ADAMTS5 antibody (GeneTex, GTX100332, 10 μg/ml) and anti-MMP13 antibody (ThermoFisher Scientific, MA5-14328, 20 μg/ml) diluted in Can Get Signal immunostaining solution (TOYOBO, Tokyo, Japan) using Vectastain ABC-AP alkaline phosphatase kit and AP substrate kit (Vector Laboratories, Burlingame, CA, United States) according to the manufacturers’ instructions. For type II and type X collagen staining, slides were pretreated with antigen retrieval reagent (Proteinase K, Dako, CA, United States) at room temperature for 10 min and blocking serum for 30 min. Then, sections were immunostained with anti-type II collagen antibody (DSHB, CIIC1, 6 μg/ml) and anti-type X collagen antibody (DSHB, X-AC9, 5 μg/ml) diluted in PBS using Vectastain Elite ABC-HRP kit and DAB substrate kit. All stainings and evaluations were performed using n = 5 each group.

Slides were pretreated with antigen-retrieval reagent (Immunoactive; Matsunami Glass Ind, Osaka, Japan) at 60 °C for 16 h, followed by blocking with 10% normal horse serum for 30 min. Then, sections were immunostained with anti-P16^INK4a antibody (abcam, ab54210; 0.1 µg/mL), anti-ADAMTS-5 antibody (GeneTex, GTX100332, 10 µg/mL), and anti-MMP13 antibody (Thermo Fisher Scientific, MA5-14,328, 20 µg/ml) diluted in Can Get Signal immunostaining solution (TOYOBO, Tokyo, Japan) using Vectastain ABC-AP alkaline phosphatase kit and AP substrate kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturers’ instructions. For type X Collagen staining, slides were pretreated with antigen-retrieval reagent (Proteinase K, Dako, CA, USA) at room temperature for 10 min, blocking serum for 30 min. Then, sections were immunostained with anti-type X Collagen antibody (DSHB, X-AC9, 5 µg/mL) diluted in PBS using VECTASTAIN Elite ABC-HRP kit and DAB substrate kit. Mouse IgG1 kappa (Thermo Fisher Scientific, 14–4714-82), mouse IgG2b kappa (14–4731-82), and rabbit IgG (14–4616-82) were used as isotype control antibodies for negative controls (Fig. S5).

After perfusion fixation with Bouin's solution, cerebelli from 0.5 to 21-day-old mice were immersed in the same solution overnight or for 1 day. Sagittal sections of 6 μm in thickness were used for morphological analyses. Hematoxylin-eosin (H&E) staining was performed with paraffin sections. Immunostaining with antibodies against c-Ret (1:150; Immuno Biological Laboratories), phosphorylated c-Ret Y1062 (1:50; Abcam), and Shh (1:100; Santa Cruz, H-160) diluted with Can Get Signal immunostaining solution (TOYOBO) was performed with paraffin and frozen sections (19 (link), 45 ). Immunostaining with antibodies against GABRA6 (1:1000; Chemicon), calbindin D28k (1:150; Chemicon), BLBP (1:200, Abcam), PAX6 (1:500, Abcam), and Ki67 (1:500, Abcam) was performed with paraffin sections. The VECTASTAIN Elite ABC kit (Vector), Envision kit/HRP (diaminobenzidine; DAB) (DAKO) with counterstaining of hematoxylin and Alexa Fluor 488-labeled donkey anti-rabbit IgG (1:1000, Invitrogen) with counterstaining of 4′, 6-diamidino-2-phenylindole (DAPI) were used. We validated the primary antibodies used in this study with no positive signals in the specimens processed under the same staining condition except for incubation without primary antibodies.