Bsb 0016

Kidney sections were immuno-stained as described previously [28 (link),31 (link),32 (link),33 (link)]. In brief, the sections were blocked with 5% normal goat serum and reacted with rabbit polyclonal anti-human Epo antibody (sc-7956, 1:10; Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by Histofine simple stain MAX-PO (414341F; Nichirei Bioscience, Tokyo, Japan). Sections were stained using DAB liquid system (BSB 0016; Bio SB, Santa Barbara, CA, USA) and counterstained with Mayer’s haematoxylin (30002; Muto Pure Chemicals, Tokyo, Japan).
Images were obtained using an optical microscope (Axio Imager M2; Carl Zeiss, Oberkochen, Germany) with a digital camera (AxioCam 506, Carl Zeiss). Captured images were analysed using an image analysing system (ZEN 2, Carl Zeiss).

Kidney sections were immuno-stained as described previously [35 (link),37 (link),38 (link),47 (link)]. In brief, the sections were blocked with 5% normal goat serum and reacted with rabbit polyclonal anti-human Epo antibody (sc-7956, 1:10; Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by Histofine Simple Stain MAX-PO (414341F; Nichirei Bioscience, Tokyo, Japan). Sections were stained using DAB liquid system (BSB 0016; Bio SB, Santa Barbara, CA, USA) and counterstained with Mayer’s hematoxylin (30002; Muto Pure Chemicals, Tokyo, Japan).
Images were obtained using an optical microscope (Axio Imager M2; Carl Zeiss, Oberkochen, Germany) with a digital camera (AxioCam 506, Carl Zeiss). Captured images were analyzed using an image analyzing system (ZEN 2, Carl Zeiss).

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).