Horseradish peroxidase labelled secondary antibody

Sections (4 μm) were obtained from formalin-fixed, paraffin-embedded normal (n = 36), CIN I (n = 50), CIN II (n = 52), CIN III (n = 44), and ISCCs (n = 61) tissues. Immunohistochemistry (IHC) staining was conducted as described previously [20 (link)] with antibodies against NOD1 (MAB7090, monoclonal mouse IgG2B clone, diluted at 1/60; R&D Systems, Minneapolis, USA), p16^INK4A (p16^INK4A (F-12): sc-1661, mouse monoclonal antibody, diluted at 1/100, Santa Cruz Biotechnology Inc., California, USA), and horseradish peroxidase-labelled secondary antibody (Maixin Biotechnology, Fuzhou, China) in accordance with manufacturer’s instructions. Color was developed with diaminobenzidine (Dako Corp, CA 95051, USA) incubated for 5–10 min at room temperature. The double immunostaining of NOD1 and p16^INK4A was performed with DouSPTM double staining kit (MaxVision, Fuzhou, China). Slides were counterstained with haematoxylin and photos were taken by light microscopy.
Staining intensity was graded according to the following criteria described in previous studies [20 (link), 21 (link)]: (−), no positive staining cells; (1+), less than 25% positive staining cells with weak intensity and focal distribution; (2+), 26–50% positive staining cells with moderate intensity and focal distribution; and (3+), more than 50% positive staining cells with strong intensity and diffuse distribution.

Sections (4 μm) were cut from FFPE tissue blocks, and IHC was conducted as described previously [25 (link)] with antibodies to OLFM4 (ab96280, rabbit anti-human polyclonal, diluted at 1/100; Abcam, Cambridge, England), ERα (Kit-0012-2, rabbit anti-human monoclonal, ready to use; Maixin Biotechnology, Fuzhou, Fujian, China), PR (Kit-0013-2, rabbit anti-human monoclonal, ready to use; Maixin Biotechnology), and horseradish peroxidase-labelled secondary antibody (Maixin Biotechnology) in accordance with manufacturer*s instructions. Colour was developed with diaminobenzidine (Dako Corp, Carpinteria, CA, USA) incubated for 5–10 min. at room temperature. Slides were counterstained with haematoxylin and examined by light microscopy.
The immunoreactive score (IRS) was used to evaluate results: IRS = staining intensity × percent of positive cells. Staining intensity was graded according to the following criteria: 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellow with brown) and 3 (strong staining, brown). The percent staining was graded according to the proportion of positive stained cells as follows: 0 for ≤5% positive cells; 1 for 6–25% positive cells; 2 for 26–50% positive cells and 3 for ≥51% positive cells. An IRS score of 4 and higher is regarded as high expression of genes.