Discovery xt staining device

Immunohistochemical staining of NaDC3 was performed on a tissue micro array (TMA) consisting of paraffin-embedded human prostate benign and cancer tissue as well as kidney tissue that was used as a positive control. The use of the archived samples was approved by the ethics committee of the Medical University Innsbruck. Tissue was counterstained with hematoxylin. NaDC3 antibody (Proteintech, Rosemont, IL, USA) was used at a concentration of 1:40 and immunohistochemistry (IHC) was performed on a Discovery-XT staining device (Ventana, Tucson, AZ, USA) using the standard IHC protocol and antigen retrieval at pH 9 (CC1). Representative images were taken with an Olympus BX53 (Olympus, Shinjuku, Japan).

For IHC staining, a tissue microarray (TMA) of 14 patients that received neoadjuvant docetaxel therapy before radical prostatectomy and 14 patients with no chemotherapy was used. The use of patient material was approved by the Ethics Committee of the Medical University of Innsbruck (study No AM 3174 including amendment 2). For detailed information about clinical data from patients, see publication of Puhr et al. (2012) (link). IHC staining was performed on a Discovery-XT staining device (Ventana) and the following specific antibody was used: anti-p300 (1:100, D8Z4E, Cell Signaling Technology). Antibody specificity was verified by Western blot and IHC staining of PC3-DR cells with p300 downregulation. For IHC, cells were embedded by coagulation in plasma clots after harvesting, transferred into a biopsy histosette, fixed in formalin, and embedded in paraffin. Importantly, cross staining of CBP was excluded by Western blot analysis.

IHC and lipofuscin staining were performed on human prostate tissue sections. Prostate cancer patients were selected from the Innsbruck Uro-biobank. The use of archived material was approved by the Ethics Committee of the Medical University of Innsbruck. Written consent was obtained from all patients and documented in the database of the University Hospital Innsbruck in agreement with statutory provisions. Two different cohorts of patients were analyzed. The control cohort comprises untreated prostate cancer patients subjected to radical prostatectomy without previous chemotherapy (n = 10); the chemotherapy group comprises prostate cancer patients who underwent neo-adjuvant chemotherapy with Taxotere (n = 10) before radical prostatectomy. Both patient groups were matched for Gleason Score and age [50 (link)].
For each prostate cancer patient, four consecutive tissue sections with 5 μm cutting thickness were obtained for analysis. H&E and p63-α-methylacyl-CoA racemase (AMACR) IHC double staining were performed for the basal characterization of prostate cancer tissue samples. IHC was carried out on a Discovery-XT staining device (Ventana, Tucson, AZ, USA), using the following antibodies: anti-P504S (AMACR) (1:100, Dako, CA, USA) and anti-p63 (4A4) (1:100; Roche, Basel, Switzerland) for prostate samples.