Fluoview ver 4.2b software

Frozen sections of A375-SM tumors were prepared as previously described [3 (link)]. Immunofluorescence analysis was performed by double staining with anti-CAII and anti-CD31 antibodies. Secondary antibodies conjugated to Alexa fluor 488 and 647 fluorochromes were used for detection followed by counterstaining with DAPI. The images were obtained with the FV10i 2.1 Viewer Software at room temperature, with a camera coupled to an objective lens with × 2.0 confocal aperture (Olympus). The Olympus FluoView ver.4.2. b software was used for image processing.
Serial sections were obtained from FFPE blocks of human RCC tumor and its normal counterparts. The sections were individually stained with anti-CAII and anti-CD31 antibodies. Immunoreactivity was visualized with HRP-linked secondary antibody (Dako) and counterstained with hematoxylin (Wako). For vessel maturity analysis, determined by the microvessel pericyte coverage index (MPI), mouse tumor FFPE sections were systematically co-stained with both anti-CD31 and anti-α-SMA antibodies in the same tissue. The anti-glut1 antibody was used to identify hypoxic tumor areas. Images were captured using a NanoZoomer 2.0-HT Slide Scanner (NanoZoomer 2.0 HT, version 2.3.27, Hamamatsu, Japan) and observed with the NanoZoomer Digital Pathology software. The antibodies used are listed in Additional file 1: Table S1.

Intracellular PpIX levels were determined using a confocal microscope or microplate reader. Cells were treated with triacsin C alone for 48 h, followed by co-culture with triacsin C and ALA for 24 h. Extracellular PpIX was removed via washing by PBS before analysis. An FV-1000D downright laser-scanning confocal microscope (Olympus, Tokyo, Japan) was used for live-cell microscope imaging. The excitation wavelength was set at 405 nm for PpIX and 635 nm for DRAQ5 (BioStatus, Loughborough, United Kingdom). The emission wavelength was set at 560–800 nm for PpIX and 655–755 nm for DRAQ5. Laser illumination was set at 3.0% power for PpIX and 5.0% power for DRAQ5. All images were acquired using a × 60 oil immersion lens. The images were analyzed using Olympus Fluoview ver. 4.2b software. A Cytation 5 microplate reader (BioTek, Winooski, VT, USA) was used for the live-cell relative quantification of intracellular PpIX content. The excitation wavelength was set at 385–425 nm for PpIX and 330–370 nm for Hoechst 33,342 (Thermo Fisher Scientific). The emission wavelength was set at 615–655 nm for PpIX and 430–470 nm for Hoechst 33,342. Fluorescence was measured at 10 points in each well.