Tomo microscope slides

Human granulation tissue samples were fixed in formaldehyde (4%) and embedded in paraffin-wax. Sections of 2.5-µm thickness were mounted on TOMO® microscope slides (Matsunami Glass, USA). Immunohistochemistry was performed using Ventana Benchmark Ultra Immunostainer (Roche, Germany).
Primary antibodies used were anti-CD31 (rabbit monoclonal, ab28364, Abcam, UK) ready-to-use without antigen retrieval and anti-CD34 (mouse monoclonal, QBEnd10, Dako, Denmark) ready-to-use with standard antigen retrieval using Ultra Cell Conditioning Solution (Ultra CC1-Ventana, Roche, Germany).

Formalin (4%)-fixed and paraffin-embedded human granulation tissue samples were sectioned at 2.5-μM thickness and mounted onto TOMO® Microscope Slides (Matsunami Glass, USA). Deparaffinization and rehydration were performed with xylol and ethanol dilution series. Antigen retrieval was performed in a water bath, by incubation in citrate buffer. The endogenous peroxidase was blocked with 3% H₂O₂ prior to antibody incubation. Primary antibody incubation was performed for 24h at 4°C using CD68 (Monoclonal Mouse, Anti-Human CD68, Clone PG-M1, Dako, Denmark) and perilipin (PLIN, Perilipin-1 (D1D8) XP® Rabbit, Cell Signaling Technology, Germany). Specimens were then treated with primary antibody enhancer, horseradish peroxidase polymer, and AEC substrate and then counterstained with hemotoxylin (Scharlau, Spain) and cover-slipped. Images were acquired with a Zeiss AXIO Imager Z2 microscope (Zeiss, Austria).

Using a microtome (Leica), 8 µm thick slices were taken from formalin-fixed paraffin-embedded (FFPE) prostate tissue blocks and mounted on Tomo microscope slides (Matsunami, Bellingham, WA, USA). Slides were then stained for IGF-II peptide using an IGF-II antibody (AbCam, Cambridge, UK) at a dilution of 1:600, on a Ventana BenchMark ULTRA™ machine (Roche, Oro Valley, AZ, USA). Slides were scored by two pathologists using a modified version of the Allred system, combining the proportion of tissue stained (a scale of 1–5) with the staining intensity (a scale of 1–3) to give a score out of 8 [55 ]. Currently, there is no standardized method of scoring prostate tissue.