Anti ep4 antibody

We detected apoptosis by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) using the ApopTag Plus Peroxidase In Situ Apoptosis Kit (Millipore, MA, USA). Kidney sections were prepared according to the procedure of the Vectastain Elite ABC kit (Vector Laboratory, CA, USA). Anti-CD3 antibody (Santa Cruz Biotechnology, CA, USA) and anti-F4/80 antibody (Abcam, Cambridge, UK) were used and revealing reaction was performed with an AEC Chromogen Kit (Immunotech, Marseille, France). All of these sections were examined in a blinded manner using light microscopy. The number of positive cells was quantified per high power field for each kidney and at least 20 fields were reviewed for each slide.
For immunofluorescence staining, sections were incubated with anti-EP4 antibody (Santa Cruz Biotechnology) and anti-aquaporin-1 (AQP-1) antibody (Millipore) overnight and then visualized with anti-rabbit Alexa Fluor-488 and -594 (Life Technologies, OR, USA). The images were captured by confocal microscopy (LSM5 Live Configuration Variotwo VRGB; Zeiss, Germany).

Kidney tissues were lysed with kinase buffer (KB; 50 mM Tris-HCl (pH 7.4), 25 mM NaF, 40 mM β-glycerol phosphate (pH 7.4), 120 mM NaCl, and 1% NP40) and then 1 mg of lysate immunoprecipitated using 1 µg of anti-EP4 antibody (Santa Cruz Biotechnology) and protein G Sepharose 4 Fast Flow (GE Healthcare, Sweden). After washing with KB without 1% NO40, immunoblotting was performed using by PGE₂ antibody (Biorbyt Ltd., UK).