Streptavidin biotin complex reagent

Sections of cartilage nodule tissue were deparaffinized and rehydrated and then treated with 3% H₂O₂. Slides were next incubated in 0.01 M citrate buffer for 20 min at 94–98°C and blocked with 5% BSA. Primary antibodies included rabbit anti-human collagen type II antibodies (1:80, Sigma-Aldrich). The staining was visualized using a microscope (Axioskop 40, Zeiss) by applying streptavidin-biotin complex reagent (Boster) and 3,3′-diaminobenzidine (DAB; Boster) after the treatment with a solution of biotinylated goat anti-rabbit IgG (Boster). Sections were treated using the same process but without incubation with a primary antibody as the negative control.

Brain tissues were fixed in 4% paraformaldehyde for 24 h. The cerebral
block was trimmed to include the CC under the impact center, immersed in
25% sucrose, and embedded in an optimal cutting tool. Serial 10-μm
thick coronal sections were obtained with a cryotome and mounted on glass
slides. The acquired brain sections were incubated in 3% hydrogen
peroxide to quench endogenous peroxidase for 15 min. After washing with 0.01 M
PBS, the sections were incubated with 5% BSA for 60 min to reduce
non-specific reactions, and treated with rabbit β-APP monoclonal antibody
at 1:4000 dilution (Abcam, ab32136, Cambridge, U.K.) at 4°C overnight.
Biotinylated goat anti-rabbit secondary antibody and streptavidin–biotin
complex reagent (Boster, Wuhan, China) were applied subsequently at 37°C
for 45 min, respectively. Positive reactions were visualized with
diaminobenzidine. The sections were dehydrated in graded alcohol, cleared in
xylene, and mounted.