Streptavidin peroxidase

Prostate tissue was paraffin embedded as previously described [8 (link)]. Four-micron thick sections paraffin embedded tissue were deparaffinized with xylene and rehydrated through a descending series of ethanol washes as previously described. KRT13 staining was tested on positive control tissue (human tonsil) and negative control tissue (human colon) in order to confirm specificity of staining and optimize antibody dilution (data not shown). Western blot analysis with KRT13⁺ A431 cell control was performed as previously described and confirmed no cross-reactivity between KRT13 antibodies (Abcam, San Francisco, CA, USA, clones ab92551, 1x10⁴ dilution and ab133340, 1x10⁴ dilution) and other cytokeratins (S2 Fig)[14 (link)]. KRT5 (BioLegend, San Diego, CA, USA, clone PRB-160-P) and KRT8 (Abcam, San Francisco, CA, USA, clone ab53280) antibodies were used in immunohistochemical analysis at 1:200 and 1:2000 dilutions, respectively. Additional immunostaining performed: PSA (DAKO, Carpentaria, CA, USA, clone A0562, 1:200); P63 (Santa Cruz, CA, USA, clone 4A4, 1:200). Both manual and automated immunostaining was performed with equivalent results. Antigen retrieval and standard immunoperoxidase procedures were used, followed by detection with streptavidin peroxidase (Biogenex, Fremont, CA, USA) per manufacturer’s instructions.

The study was approved by the National Taiwan University Hospital Ethics Committee. Paraffin-embedded tumor blocks were dewaxed and pretreated in citric acid buffer solution (pH 6.0) (for ki67 staining) or boric acid solution (for G9a staining) by microwave boiling for 15 min to retrieve the antigens. Sections were then incubated with 1% H₂O₂ in methanol for 15 min at room temperature to block endogenous peroxidase; they were then blocked with 3% bovine serum albumin for 1 h at room temperature. Staining was performed at 4°C overnight with the indicated antibodies, anti-G9a (R&D Systems, Minneapolis, MN, 1:100) and Ki-67 (GeneTex, 1:100). Sections were incubated with immunoglobulin G (IgG)-biotinylated secondary antibodies (BioGenex) for 40 min then incubated with streptavidin peroxidase (BioGenex) for 30 min at room temperature. Visualization was achieved using 0.03% diaminobenzidine tetrahydrochloride substrate for 3 min, and then sections were counterstained with Mayer’s hematoxylin and mounted. We classified staining intensity having as low (negative and focal expression less than 20% of tumor cells) and high (diffuse expression in more than 20% of tumor cells) expression.