Bz x analyzer

Digital images of over 30 glomeruli or over 30 tubulointerstitial areas randomly selected from each mouse were obtained at 400× magnification using an All-in-One Fluorescence Microscope BZ-X710 (Keyence, Osaka, Japan). The size and number of total cells in each glomerulus were determined using PAS-stained sections. The number of B220⁺ B-cells, CD3⁺ T-cells, Iba1⁺ macrophages, and CD34⁺ capillaries observed in the digital images of glomeruli were counted using immunohistochemical sections and a BZ-X Analyzer (Keyence). Further, glomerular damage was semi-quantitatively scored according to methods described by Ichii et al. [23 (link)]. For TILs, the numbers of B220⁺ B-cells, CD3⁺ T-cells and IL-1F6/IL-36α⁺ damaged tubules throughout the cortex were counted. Additionally, CD34⁺ capillaries, Iba1⁺ macrophages, and αSMA⁺ reaction areas in the tubulointerstitium were counted using immunohistochemical sections and a BZ-X Analyzer (Keyence), based on digital images of the renal cortex.

Liver tissue samples were collected at 6 h after the injection of TNF-α or ConA, fixed in 10% formalin and embedded in paraffin. Immunostaining was performed in the same way as human pathological examination. TF antibody (ab151748; Abcam), hypoxia-inducible factor (HIF)-1α antibodies (NB100-479; Novus Biologicals), HIF-2α antibodies (NB100-122; Novus Biologicals), LDH-V antibody (ab85472; Abcam) and vascular endothelial growth factor (VEGF)-A antibody (ab183100; Abcam) were used as the first antibody. Secondary goat anti-rabbit antibodies (Histofine Simple Stain kit; Nichirei Bioscience) was applied. The sections were visualized under a Keyence BZ-X700 microscope (Keyence). Positive areas in five randomly selected microscopic fields (x40 magnification) per section were measured using analysis software (BZ-X analyzer, Keyence, Osaka, Japan). For pimonidazole staining, Hypoxyprobe Omni kit (Abcam) was used according to the manufacturer's protocol (21 (link)). Positive areas in five randomly selected microscopic fields (magnification, x40) per section were measured using analysis software (BZ-X analyzer; Keyence) and the mean percentage of positive area was calculated.

The superoxide production of aortic rings and PVAT were assessed with dihydroethidium (DHE) (D11347, Invitrogen, MA, USA) as previously described [20 (link)]. The frozen sections of aortic rings both without and with PVAT were cut into 10 µm thick sections and placed on glass slides. The samples were incubated at room temperature for 30 min with DHE (2 × 10⁻⁶ mol/L) and protected from light. Images of the samples were observed using a microscopic system (BZ-X710, Keyence, Osaka, Japan) with an excitation wavelength of 540 nm and an emission wavelength of 605 nm. The fluorescence intensity of the DHE staining was measured using a BZX analyzer (Keyence, Osaka, Japan). In addition, we employed the tempol (176141, Sigma-Aldrich, MO, USA), a superoxide scavenger, in order to test the contribution of superoxide production for the aorta and PVAT. We incubated the aortic rings both without and with PVAT, with tempol (10⁻⁴ mol/L) for 1 h, and then made the slides of these samples with tempol. The samples were incubated at room temperature for 30 min with DHE (2 × 10⁻⁶ mol/L) and protected from light. Images of the samples were observed using a microscopic system (BZ-X710, Keyence, Osaka, Japan) with an excitation wavelength of 540 nm and an emission wavelength of 605 nm. The fluorescence intensity of the DHE staining was measured using a BZX analyzer (Keyence, Osaka, Japan).