Vanox t ah 2 light microscope

Immunohistochemistry stained sections were examined using an Olympus Vanox-T AH-2 light microscope (Olympus, Tokyo, Japan). The same magnification (microscope objective) was used to record all images in a particular series. Both the field limiting and contrast apertures were kept at the fully open position during (digital) photography to avoid any variability in reproducing aperture settings. Images were recorded with a Spot RTCCD camera (Diagnostic Instruments, Sterling Heights, MI, USA) in color mode, using full (1600× 1200 bit) resolution at 8-bit depth for each (RGB) color component. The photographs were taken with identical exposure settings. A flat field image was obtained at the beginning of each microscopy session and then used throughout the session to correct for uneven illumination. The white balance was adjusted for each slide. Recorded images were stored in color mode as uncompressed files in tagged image file format. The subsequent digital images were analyzed using the built-in functions of ImagePro Plus image analysis software 6.0 (Media Cybernetics, Silver Spring, MD, USA) by three pathologists simultaneously. Five fields of view were chosen stochastically by each pathologist. The procedure for determining the immunostaining intensity was as previously described[40 (link)].

Immunohistochemistry (IHC) was conducted to measure SRC and RhoA expression as described previously (Du et al., 2017). In brief, paraffin‐embedded tissue was pretreated at 65 °C for 2 h, followed by deparaffinization using standard procedures. Antigen retrieval was performed in antigen retrieval solution before application of primary antibodies. Slides were then incubated for 2 h at room temperature with the secondary antibody conjugated with horseradish peroxidase (HRP). HRP activity was detected using the Histostain‐plus kit (Invitrogen) in accordance with the manufacturer's instructions. Evaluation of IHC staining was performed as described previously (Waltregny et al., 1998). In brief, IHC‐stained sections were examined using a Vanox‐T AH‐2 light microscope (Olympus, Tokyo, Japan). The same magnification (microscope objective) was used to record all images in a particular series. Both the field limiting and contrast apertures were kept at the fully open position during (digital) photography to avoid any variability in reproducing aperture settings. Furthermore, the IHC‐stained sections were reviewed by two independent observers unbiased without the knowledge of clinical outcome.