Envision flex detection kit

Immunohistochemistry was performed on archival paraffin embedded human tissue obtained from normal colon, duodenum, lung, thymus, liver, spleen. Six needle biopsies from the prostate gland were included. Two needle biopsies contained only malignant tissue, while the remaining four contained both benign tissue and adenocarcinoma. Gleason score was between 6 and 9. Furthermore, different subtypes of colonic adenocarcinomas and colonic adenomas/precursor lesions were included. Tissue material had been fixed in buffered formalin and embedded in paraffin. Three micron sections were made. Immunochemical staining was done using the following murine antibodies as primary antibody OLFM4 #40 (0.48 μg/ml), OLFM4 #49 (0.70 μg/ml). The sections were pre‐treated in PT Link (Dako, Glostrup, Denmark) using high pH target retrieval solution (DM828; Dako) and stained in a DakoLink 48 (Dako) utilizing the Envision Flex+ detection kit (K8002; Dako). Primary antibodies were diluted in Antibody Diluent (DM830; Dako) and incubated for 20 min. The sections were counterstained with haematoxylin (Dako) and analysed using an Olympus BX51 microscope (PlanApo 20x/0.70 objective) with an Olympus UC30 camera and the Olympus cellSens software package (Olympus, Ballerup, Denmark).

For immunofluorescence, cells were fixed with 4% PFA for 15 min at RT. Afterward, cells were permeabilized with 0.2% Triton-X100 for 3 min at RT and blocked with 3% BSA (Sigma) for 30 min. The primary antibody Anti-H2A-X (Ser139) clone JBW301 (Millipore #05-636) was diluted (1:200) in 0.3% BSA and incubated overnight at 4 °C. Next, the secondary antibody labeled with Alexa 488 (Molecular Probes, Eugene, OR, USA) at a 1:1000 dilution was added in 0.3% BSA in darkness at RT together with Hoechst (Sigma) to stain cell nuclei. Epifluorescence images were acquired in an inverted Olympus IX71 confocal microscope.
For IHC, sample sections of 3 μm were blocked for endogenous peroxidase and incubated with primary antibody Ki67 (Ready to Use (RTU), Dako, Glostrup, Denmark). The reaction was visualized with the EnVision FLEX Detection Kit (DAKO). Sections were counterstained with haematoxylin.
For determination of cellular DNA content, cells were trypsinized, fixed in 70% ethanol, and incubated for 30 min at 37 °C in 1× saline sodium citrate containing 50 µg/mL RNase A and 50 µg/mL propidium iodide prior to analysis by flow cytometry.

From the Tumorbank of the Antwerp University Hospital (Belgium), tissue of a giant cell tumor of bone and an osteoblastoma were obtained. Tissue specimens were fixed in 4% formaldehyde and paraffin embedded on a routine basis. Five μm-thick sections were subjected to heat-induced antigen retrieval by incubation in 10mM citrate buffer (pH 6.0) for 20 minutes at 97°C. Subsequently, endogenous peroxidase activity was quenched by incubating the slides in peroxidase blocking buffer (DAKO) for 10 minutes. Incubation with primary anti-human ZIP14 antibody (PA5-21077, Thermo Fisher Scientific, 1:200 dilution) was performed at room temperature for 1 hour. Bound antibody was detected with the Envision FLEX+ detection kit (DAKO) using 3,3’-diaminobenzidine chromogen solution (DAKO). A negative control, using a rabbit IgG isotype control (10500C, Thermo Fisher Scientific, 11.2ng/μL) was included in each staining run and did not show positive expression in osteoblasts or giant cells (S7 Fig). Sections were counterstained with haematoxylin, dehydrated and mounted.