In Situ Hybridization of Renal Markers

ISH was performed, as described previously [5 (link),27 (link)]. In brief, total RNA from the mouse kidneys (636612; BD Bioscieces Clontech) was reverse-transcribed with an RNA PCR kit (AMV), ver. 3.0 (RR019; Takara), and the cRNA probe for Epo (GenBank accession no. NM_007942), HIF2α (GenBank accession no. NM_010137), or PHD2 (GenBank accession no. NM_053207) was generated with T7 promoter region-tailed PCR primers. The hybridized sections were successively treated with 0.1% avidin, 0.01% biotin solution, 0.5% casein/TBS, horseradish peroxidase (HRP)-conjugated sheep anti-DIG F (ab′) fragment antibody (11207733910; Roche Diagnostics, Basel, Switzerland), biotinylated tyramide solution, and HRP-conjugated streptavidin (P0397; DakoCytomation, Glostrup, Denmark). Sections were stained using the DAB liquid system (BSB 0016; Bio SB, Santa Barbara, CA, USA) and Mayer’s hematoxylin (30002; Muto Pure Chemicals, Tokyo, Japan).