Bz x analyzer software

To evaluate migration ability of CDCs, scratch assay was performed as described previously42 (link). In 24-well culture dishes at high confluence, scratches were created using 1000 μl tips. Phase contrast images of the scratches were acquired using BZ-X710 All-in-One fluorescence microscope (KEYENCE) at 0 h and 12 h after incubation. The area of wounds was measured by using BZ-X Analyzer software (KEYENCE), and wound closure rates were calculated.

Regular epifluorescence images were acquired on a Keyence BZ-9000 inverted epifluorescence microscope equipped with an RGB and monochrome camera (Keyence, Osaka, Japan). Either the entire coronal hemibrain slice or particular brain regions were scanned using 10x magnification and stitched in Keyence BZ-X Analyzer software (V1.3.0.3).

To examine the histological and pathological changes of DHW-221 in Taxol-resistant lung neoplasm, HE staining and immunohistochemistry (IHC) were performed. All tissues were fixed with 4% paraformaldehyde, dehydrated in a graded ethanol solution (70%, 80%, 90%, 95%, and 100%, v/v), incubated in xylene until transparent, and embedded in paraffin. Subsequently, the tissues were cut into approximately 5-µm-thick sections for further study. For pathological examination, paraffin sections were sequentially deparaffinized in a xylene (xylene I, xylene II, and xylene III) and graded alcohol solution (90%, 80%, and 70%, v/v). Paraffin sections were stained with hematoxylin and eosin (HE) to distinguish tumorous metastatic foci and normal tissues. For IHC, the paraffin sections were incubated with the following primary antibodies: anti-Ki67, anti-FOXO3a, and anti-p-FOXO3a. The sections were then incubated with the appropriate secondary antibody according to the DAB kit. Finally, images were acquired using a Keyence BZ-X700 microscope and BZ-X analyzer software (Osaka, Japan). Brown granules were considered as positive staining in the cytoplasm or nucleus.

Conventional methods for measuring RF use Masson trichrome staining of randomly obtained magnified fields, followed by image analysis with Image J software. However, because of possible biases in selecting fields, a Biorevo BZ-9000 microscope (Keyence, Osaka, Japan) and BZ-X analyzer software (Keyence) were used. After automated capture of 40x magnified images over the entire tissue sample, a single high-resolution image of the whole kidney was created seamlessly (Supplementary Fig. S6a,b). Each whole kidney image was dissected into four portions (CO, OS, IS, and IM), and the renal fibrosis in each portion was quantified by Micro Cell Count software (Keyence) using common parameters (Supplementary Fig. S6c–f). Sirius Red staining was used instead of Masson trichrome staining, because the former is more specific for collagen types I and III³⁵ . DAB based immunohistochemistry (collagen type I or α-SMA) was not used, because the software cannot be applied to the heterogeneous background staining observed in DAB based immunohistochemistry.

Formalin‐fixed, paraffin‐embedded mid‐ventricular sections were stained with periodic acid–Schiff for cardiomyocyte cross‐sectional area (CSA), Masson's trichrome for fibrosis, and rabbit‐anti‐mouse CD31 (1:50, Cell Signaling Technologies, #77699) for capillary density. Cardiomyocyte CSAs were measured in three to five random sections (40–60 cells/section, ~200 cells/heart), which were averaged to represent a single data point for each heart. Capillary density was quantified by dividing the number of CD31+ cells by the area of randomly selected sections. Three to five sections were measured per heart and averaged to represent a single data point. Given the extensive variability in fibrosis distribution throughout the heart, BZ‐X Analyzer software (Keyence) was used to quantify fibrosis in full mid‐ventricular sections. Percent fibrosis was calculated as the ratio of fibrotic area to total tissue area. Measurements from two sections were averaged to represent a single data point for each heart. Quantitative histologic analyses were done in a blinded fashion.