Ventana discovery staining system

The cryo-section were washed for 10 min. in Ventana Reaction Buffer (Roche Diagnostics®, Rotkreuz, Switzerland) and fluorescence staining was performed using a Ventana discovery staining system (Roche Diagnostics®, Rotkreuz, Switzerland) using the standard protocol for cryo sections (frozen 402). To show the preservation of the nervous tissue, a concentration of 1:100 for beta-3Tubulin (Abcam® plc., Cambridge, United Kingdom, cat.-nr. ab52623) antibody was selected. The condition of IHC as well as OHC were illustrated with a Myosin-VIIa primary antibody (Proteus Biosciences® Inc., Ramona, CA, United States, cat.-nr. 25–6,790).
The apoptotic activity in the tissue was visualized with a primary antibody for CC3 at a concentration of 1:400 (Cell Signaling®, Leiden, Netherlands, lot. 47/9661S), BAX 1:10 (Santa Cruz Biotechnology® Inc., Dallas, Texas, United States, lot. B2912) and as antagonist BCL2 1:50 (Santa Cruz Biotechnology® Inc., Dallas, Texas, United States, lot. C0805 and lot. C0207) was utilized. As secondary antibody Alexa Rabbit 594 (Life Technologies™, InVitrogen® Inc., Darmstadt, Germany, ref. A21207, lot. 2,313,074) was selected in a concentration of 1:200.

Tissue specimens were immunostained using the Ventana Discovery Staining System (Roche, Basel, Switzerland) and the DISCOVERY ChromoMap DAB Kit (Roche) according to the manufacturer's instructions. The tissue sections were preheated with CC1 (pH 9.0, Roche) for 30 min at 100 °C. They were then incubated with the mouse monoclonal anti‐p‐hTERT antibody (1:500 dilution) or Ki67 (MIB1, 1:100 dilution; Agilent Technologies, Inc) for 12 h at 25 °C. Incubation with OmniMap anti‐mouse HRP‐conjugated multimer secondary antibody (Roche) was performed for 32 min at 25 °C. We performed immunohistochemical staining of mouse IgG1 (isotype control; Abcam, Cambridge, UK) under the same conditions as p‐hTERT and Ki67 staining, and confirmed negative staining. The proportion of cancer cells with positively stained nuclei was analyzed at ×200 by the authors (YMa, JY, KY, or TY) [16 (link)]. The percentage of p‐hTERT and Ki67 expression was scored every 10% as follows: 0–9%, 0; 10–19%, 10; 20–29%, 20; and so on.