Micro cell count software

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.

Cells were incubated with one of the following primary antibodies diluted in 0.1% BSA/PBS: anti-fibroblast marker (ER-TR7) (sc73355, Santa Cruz Biotechnology, TX, USA, 1:100), anti-CD90 (ab3105, Abcam, Cambridge, UK, 1:300), or anti-FABP4 (EPR3579) (ab92501, Abcam, 1:1000). After washing with PBS/0.1% Tween 20 (Wako Pure Chemical Industries, Japan), sections were incubated with Alexa Fluor 488-conjugated (Invitrogen, CA, USA, 1:500) or TSA Fluorescein System (NEL 702, PerkinElmer Life Sciences Inc, MA, USA) secondary antibodies for 1 h at room temperature. After counter-staining with Hoechst 33258 (94403, Sigma-Aldrich, MO, USA) to visualize nuclei, images were obtained with BZ9000 (Keyence, Japan). Stained cells were observed under a BioRevo BZ-9000 microscope (Keyence), and the number of fluorescence-positive cells was counted using the MicroCell Count software (Keyence).