Optimizing Immunohistochemical Prostate Tissue Analysis

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.