Dako omnis staining system

For construction of the tissue microarray (TMA), one tissue core (diameter 1 mm) of a representative tumor area was taken from a “donor” block and was arranged in a new “recipient” block using the TMA Grandmaster (3DHISTECH, Budapest, Hungary).
Hematoxylin and eosin stain slides were automatically developed on a Tissue-Tek Prisma Plus staining device (Sakura Finetek, Torrance, CA, United States). All IHC-analyses were conducted using the DAKO Omnis staining system (Agilent, Santa Clara, CA, United States) with the DAKO FLEX-Envision Kit (Agilent) according to manufacturer’s instruction. We performed staining of CK5/6 (Clone: D5/16 B4; ready-to-use kit; Dako/Agilent, Santa Clara, CA, United States) and GATA3 (Clone: L50-823; ready-to-use kit; Cell Marque, Rocklin, CA, United States). IHC “double negative” was defined as negative for expression of CK5/6 and GATA3 and “double positive” was defined as positive for expression of CK5/6 as well as GATA3.
Stained slides were scanned with the Pannoramic slide scanner (3DHISTECH, Budapest, Hungary). The quantitative analysis of IHC was annotated by two genitourinary pathologists. TMA cores with either absence of representative tumor tissue or presence of staining artifacts were excluded from the analysis.

All cases were stained with an antibody against p16. Briefly, freshly cut 1 µm thick paraffin sections were stained using CINtec® (Roche, Basel, Switzerland) according to manufacturers’ instructions. Immunohistochemistry was performed using the DAKO FLEX-Envision Kit (Agilent, Santa Clara, CA, US) and the fully automated DAKO Omnis staining system (Agilent, Santa Clara, CA, US) according to manufacturer´s instructions. Epitope retrieval was generated at pH6 and 97 °C. Epitope staining was applied for 30 min. Epitope visualization was done by DAKO EnVision™ FLEX DAB + Substrate Chromogen System (Agilent, Santa Clara, CA, US). DAKO haematoxylin solution (Agilent, Santa Clara, CA, US) was used for nuclear counterstaining. Positive staining was noted in the case of block like positive staining. Mosaic staining pattern was noted as negative.

Paraffin sections were stained with the antibodies p53 and gammaH2AX. For staining, the DAKO FLEX-Envision Kit (Agilent, Santa Clara, CA, US) and the fully automated DAKO Omnis staining system or manual procedure (Agilent, Santa Clara, CA, US) were applied according to manufacturer´s instructions. The antibodies used were Anti- Anti-phospho-Histone H2A.X (gammaH2AX) (Ser139) (ZRB05636, dilution 1:100, Merck KgA, Darmstadt, Germany) applied for 30 min after 95 °C epitope retrieval in pH9 and anti-Human p53 Protein (DO-7, ready to use dilution, Dako, Agilent, Santa Clara, CA, US) applied for 20 min after epitope retrieval in pH9. Epitope visualization was performed using DAKO EnVision™ FLEX DAB + Substrate Chromogen System (Agilent, Santa Clara, CA, US). Nuclear counterstain was done using DAKO hematoxylin solution (Agilent, Santa Clara, CA, US). Slides were finally digitized using Pannoramic Scan II (3D Histech, Budapest, Hungary.