Apoptagplus peroxidase in situ detection kit

Cell death was analyzed on paraffin tissue sections using the ApopTagPlus Peroxidase in situ detection kit (Millipore). Cell proliferation and transport protein expression was evaluated by immunohistochemistry using mouse anti-BrdU (Dako, Hamburg, Germany, clone Bu20a, dilution: 50×), goat anti-CNT1 (Santa Cruz Biotechnology, code sc48457, dilution: 200×), rabbit anti-CNT3 (Santa Cruz Biotechnology, code sc134529, dilution: 200×), rabbit anti-ENT1 (Abcam, code ab135756, dilution: 500×), rabbit anti-PMAT (Antikörper-online, code ABIN754948, dilution: 300×), rabbit-anti-OCT1/2 (Antikörper-online, code ABIN754948, dilution: 800×). The Universal LSAB+ Kit/HRP (Dako) was used as secondary antibody.

After prostatectomy, tissues were fixed in 10% neutral-buffered formalin, cut into slices of 0.5 cm thickness and embedded in paraffin. Three μm whole-mount sections were cut, mounted on slides and stained with haematoxylin and eosin (H.-E.). The percentage of tumor tissue showing therapy-associated regressive changes was estimated according to Srigley et al. 2012 [28 (link)]. Representative tumor regions regarding growth patterns, grading and tumor regression were selected of each specimen and stained with the Apoptag^® Plus Peroxidase in situ detection kit (Millipore, S7101) to determine the apoptotic rate according to the TUNEL method using diaminobenzidine (DAB) as detection system. Counterstaining was performed with haematoxylin. Stained sections were visualized under bright field microscope and the number of TUNEL positive tumor cells per high power field (HPF) was determined as the average of at least 10 HPFs (prostatectomy specimen of the therapy and control group) or 5 HPFs (pretherapeutic biopsies of the therapy group), respectively.