Dab horseradish peroxidase color development kit

Limbs were fixed in 4% paraformaldehyde for 24 h and decalcified in 10% EDTA for 3 days. Paraffin sections (4-µm) were obtained and stained with Hematoxylin and Eosin (H&E), safranin O and toluidine blue. Mean values of heights of the reserve, proliferative, hypertrophic zones, and the total growth plate were calculated from measurements taken at 3 positions across the proximal tibia growth plates using ImageJ version 1.44p software. IHC/Immunocytochemistry (ICC) was performed with Hsitostain-Plus kit (ZSGB-BIO, China). Primary antibodies included: collagen II (Sigma, USA), sox 9 (Abcam, UK), collagen X (Abcam, UK), and activated-caspase 3 (Bioworld, USA). Detection was conducted with a DAB horseradish peroxidase color development kit (ZSGB-BIO, China). Semi quantitative analysis of IHC images through integrated optical density (IOD) was taken using Image-Pro Plus version 6.0.0.260 software.

Nucleus pulposus tissues were washed with PBS and then fixed in 10% paraformaldehyde for 48 h. The tissues were then embedded in paraffin and sectioned at 5 μm. The sections were then deparaffinized, and antigen retrieval was performed by immersing the samples in 95°C EDTA antigen retrieval solution (pH = 6.0, Solarbio) for 30 min. Endogenous peroxidase activity was abolished by 3% hydrogen peroxide treatment for 15 min. Sections were then blocked in 2% normal goat serum for 30 min. Next, sections were incubated with primary antibodies overnight at 4°C. Sections were incubated in HRP-conjugated secondary antibody for 30 min incubation at room temperature. A DAB Horseradish Peroxidase Color Development Kit (ZSGB-BIO) was used for detection. Immunostaining evaluations were performed independently by experimenters blinded to the sample identity. Sections were then immediately washed with tap water, counterstained in hematoxylin for 20 s, and washed again with tap water before dehydration and mounting.

The immunoreactive expression of MasR in the lung was observed by immunohistochemistry staining. The 4-µm thick paraffin sections were cut and stored at 58°C for 3 h after dewaxing in xylene. MasR (1 : 500) primary antibody was then added and incubated overnight at 4°C. Thereafter, the sections were flushed with PBS three times and incubated with secondary antibodies (1 : 20000 dilution) for 20 min. This was followed by visualization with 3,3′-diaminobenzidine (DAB) horseradish peroxidase color development kit (Zsbio, No. ZLI-9018). Immunohistochemical positive signals were stained brown in the cell membrane [21 (link)], and the optical density (OD) was analyzed using Image Pro Plus 6.0 software.

The evaluation of PinX1 staining was performed in a blinded, independent manner by two pathologists. Immunohistochemistry (IHC) was performed by standard operating procedures, and a semiquantitative scoring method according to intensity (no, very weak, intermediate, and strong staining); no staining and very weak staining were considered “negative,” whereas intermediate and strong staining were considered “positive.” A primary polyclonal anti-PinX1 antibody (1 : 200; Sigma, St. Louis, MO, USA) and polymer peroxidase-labelled secondary antibody (ZSGB-Bio, Beijing, China) were used in IHC, followed by staining using a DAB Horseradish Peroxidase Color Development Kit (ZSGB-Bio, Beijing, China) [24 (link)].

Phycoerythrin (R-PE, A_max/A_280nm > 5.0) was purchased from Hongrui Biotech Company (Pan’an County, Zhejiang, China). MTT, trypsin, PI, RNase A, Tween-20, glycine, acrylamide, methylene diacrylamide and Tris were purchased from the Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 cell culture medium and trypsin were purchased from Gibco (Grand Island, NY, USA). Doxorubicin hydrochloride was purchased from Pfizer Pharmaceuticals Limited (Brooklyn, NY, USA). Fetal calf serum (FCS) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Hoechst33258, penicillin-streptomycin, cell lysis buffer, and tetramethylethylenediamine (TEMED) were obtained from the Beyotime Institute of Biotechnology (Shanghai, China). DAB Horseradish Peroxidase Color Development Kit was purchased from ZSGB-BIO (Beijing, China). All other chemicals and solvents used were the highest purity grade.

Paraffin sections of the cartilage pellets and mouse knee joints were prepared for IHC experiments using the Histostain‐Plus Kit (ZSGB‐BIO, China). The antibodies included anti‐COL2A1 (1:200), anti‐SOX9 (1:500), anti‐MMP9 (1:200) and anti‐MMP13 (1:1000) antibodies. Detection was performed using a DAB Horseradish Peroxidase Color Development Kit (ZSGB‐BIO, China), and staining intensity was scored according to the previously published article.²⁴