Aida 2.1 image analysis software

Frozen renal tissue was homogenized, protein samples were prepared as described [57 (link)] and separated on a denaturing SDS-PAGE gel [58 (link)]. After electrophoresis, the gels were electroblotted onto PVDF membranes (Hybond-P, GE Amersham, Munich, Germany), blocked with Rotiblock (Roth, Karlsruhe, Germany) for 1 h and incubated overnight with a primary antibody to chemerin. Protein bands were visualized with secondary horseradish peroxidase-conjugated IgG antibodies (Santa Cruz Biotechnology, 1:50,000), using the Pierce ECL+ system (Thermo Fisher Scientific, Waltham, MA, USA). Blots were quantified using a luminescent imager (LAS-1000, Fujifilm, Berlin, Germany) and Aida 2.1 image analysis software (Raytest, Berlin, Germany). Loading of the blot was quantified by Amido Black staining solution (Sigma, Taufkirchen, Germany).

Frozen renal tissue was homogenized, protein samples were prepared as described (Menendez-Castro et al., 2014 (link)) and separated on a denaturing SDS-PAGE gel (Laemmli, 1970 (link)). After electrophoresis, the gels were electroblotted onto PVDF membranes (Hybond-P, GE Amersham, Munich, Germany), blocked with Rotiblock (Roth, Karlsruhe, Germany) for 1 h and incubated overnight with a primary antibody to VEGF-A (ab46154, Abcam, 1:1000). Protein bands were visualized with secondary horseradish peroxidase-conjugated IgG antibodies (Santa Cruz Biotechnology, 1:50000), using the ECL system (GE Amersham, Freiburg, Germany). Blots were quantified using a luminescent imager (LAS-1000, Fujifilm, Berlin, Germany) and an Aida 2.1 image analysis software (Raytest, Berlin, Germany). Loading of the blot was quantified by reprobing with an antibody to tubulin (Sigma, Taufkirchen, Germany, 1:10000).