Streptavidin biotin peroxidase complex

To track MSC, EGFP renal expression was studied by immunohistochemistry as already described 35. Briefly, 3‐μm‐thick sections of paraffin embedded tissue were collected on poly‐L‐lysine‐coated slides (Dako, Glostrup, Denmark). These were dewaxed in xylol, passed in a decreasing series of alcohol and rehydrated with distilled water. Endogenous peroxidase was blocked with H₂O₂/methanol 3.7% vol/vol for 10 min. followed by H₂O₂. After three washings in PBS, the sections underwent microwave antigen retrieval, then were exposed overnight at 4°C to monoclonal mouse anti‐green fluorescent protein antibody IgG1 (Chemicon International, Billerica, MA, USA). After three washings in PBS, the immunocomplex was visualized with the biotin–streptavidin–peroxidase complex and 3,3‐diaminobenzidine (Dako). Sections were counterstained with Harris haematoxylin. Negative controls included both omission of the primary Ab and substitution of IgG for primary antibodies. Kidney sections of SD‐EGFP were used as positive controls. We counted EGFP‐positive cells/HPF (×400) in 10 renal sections per kidney.

Paraffin-embedded tissue sections were mounted on poly-l-lysine-coated slides (Dako, Carpinteria, CA, USA), dewaxed in xylol, cleared in a decreasing series of alcohol, and rehydrated with distilled water. Endogenous peroxidase was blocked by treatment with H₂O₂ (3.7% vol) followed by H₂O for 15 min. After 3 washes in 150 mM PBS, the sections underwent microwave antigen retrieval, and were exposed overnight at 4 °C to monoclonal mouse anti-PCNA antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA).
After another 3 washes in PBS, the immunocomplex was visualized with biotin-streptavidin-peroxidase complex and 3,3-diaminobenzidine (Dako, Glostrup, Denmark). The sections were lightly counter-stained with Harris hematoxylin. Negative controls were established by omitting the primary antibody and substituting immunoglobulin G for the primary antibodies.
We analyzed 10 non-consecutive sections from each immunostained kidney. The images were captured using a Nikon Eclipse E200 microscope (Amsterdam, The Netherlands) connected to a charge-coupled device (CCD) camera and ImageJ, an image-analysis software (NIH, Bethesda, MD, USA).
The tubular cell proliferation index was defined as the ratio between the nuclei expressing PCNA and the total nuclei in each tubule in every field analyzed (magnification, ×40).

High density tissue microarrays were obtained from Biomax (OR601a). The presence of agrin was analyzed in 10 cancer-adjacent normal tissues and in 47 primary oral squamous cell carcinomas by immunohistochemistry using the streptavidin-biotin peroxidase complex (Dako). Protein quantification was assessed with the aid of Aperio Scanscope CS Slide Scanner and the Pixel Count V9 algorithm software (Aperio Technologies, Vista, CA; USA). By using specific input parameters, the percentage of cytoplasm positivity was calculated and classified as weak, moderate and strong, according to its staining intensity. Each category received an intensity score, 1 to weak, 2 to moderate and 3 to strong staining. The final score of each tissue sample was calculated as the sum of the percentage of each category multiplied by their respective intensity scores as the following formula: Score = (%weak×1)+(%moderate×2)+(%strong×3). Clinical pathological data, such as sex, age, anatomical site of the primary tumor, clinical stage and histopathological grade were collected from patient’s charts and showed in S2 Table.