Mayer s hematoxylin and eosin y solution

After imaging, the matrix layer was washed off with 80 vol% aqueous ethanol (EtOH purissimum, Roth, Germany) and stained according to the hematoxylin and eosin (H&E) staining protocol (Mayer's Hematoxylin and Eosin-Y solution, Sigma-Aldrich; m- and p-Xylol for analysis, Merck; Eukitt quick hardening mounting medium for microscopy, Honeywell-Fluka, USA). Optical images (250 × magnification) were recorded after the mounting medium has dried, using a digital light microscope (VHX-5000, Keyence).

LCM was described in^{58 (link)}. Briefly, OCT embedded mouse kidney sections were stained using Mayer’s hematoxylin and eosin Y solution (Sigma). LCM was performed using the Leica LMD laser capture microdissection system (Leica Microsystems GmbH, Wetzlar, Germany). A total of ≈200 tubular cuts were captured for each sample. Total RNA was isolated using the RNeasy micro RNA isolation kit (Qiagen) and reverse-transcribed using iScriptTM reverse transcription (Bio-Rad) and qPCR was performed using QuantiFast SYBR Green (Qiagen) with the primers listed in Table 1.

Tissue samples were fixed with 10% formalin and dehydrated by various concentrations of alcohol, according to our previous study.³⁴ After embedding in paraffin, tissues were cut into sections of approximately 5 μm. In the process of hematoxylin and eosin (H&E) staining, the slides were incubated in Mayer's hematoxylin and eosin Y solution (Sigma‐Aldrich), and then the images were taken using an optical microscope (Olympus, Tokyo, Japan). For immunohistochemical staining (IHC), the slides were incubated with antibodies against Ki‐67 (Cell Signaling Technology, MA) at 4°C overnight, and then incubated with secondary antibodies at 20‐25°C for 2 hours, followed by mounting with mounting medium (Dako, Glostrup, Denmark). Images were taken using an optical microscope (Olympus). For immunofluorescence analysis, the slides were incubated with antibodies against XIAP or survivin (Abclonal, MA) at 4°C overnight, and then incubated with secondary antibodies Alexa Fluor 488 (Invitrogen), Alexa Fluor 568 (Invitrogen) at 20‐25°C for 2 hours or DAPI (Invitrogen), followed by mounting with fluorescent mounting medium (Dako). Images were captured by using a confocal microscope (Carl Zeiss LSM 780; Jena, Germany).