Quantifying HIF-1α and HIF-2α in Kidney

Transverse paraffin-embedded kidney sections (7 μm) were immunostained for HIF1α as described (23 (link)) with primary antibody binding detected using biotinylated rabbit anti-mouse (Dako, Carpinteria, CA, USA). Contiguous sections were also immunostained for HIF2α by replacing the anti-HIF1α antibody with an anti-HIF2α antibody (Novus Biologicals, Cambridge, United Kingdom). Isotype-matched IgG was used as a negative control. Images of immunostained sections were captured in a blinded fashion using a light microscope and mounted camera (M165FC and DFC310FX; Leica, Newcastle upon Tyne, United Kingdom). Positive staining was quantified as previously described (24 (link), 25 (link)) and expressed as average percentage of transverse tissue area throughout the length of the tissue.

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Ashmore T., Fernandez B.O., Evans C.E., Huang Y., Branco-Price C., Griffin J.L., Johnson R.S., Feelisch M, & Murray A.J. (2014). Suppression of erythropoiesis by dietary nitrate. The FASEB Journal, 29(3), 1102-1112.

Publication 2014

biotinylated rabbit anti-mouse anti-HIF2α antibody M165FC DFC310FX

Anti antibody Antibody Biologicals Hif1α Isotype Kidney Light microscope Mouse Novus Paraffin Rabbit Tissue

Corresponding Organization : University of Cambridge

Other organizations : University of Southampton

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Variable analysis

independent variables

Immunostaining for HIF1α
Immunostaining for HIF2α

dependent variables

Positive staining for HIF1α
Positive staining for HIF2α

control variables

Transverse paraffin-embedded kidney sections (7 μm)
Biotinylated rabbit anti-mouse antibody (for detecting HIF1α primary antibody)
Anti-HIF2α antibody (for detecting HIF2α)

controls

Positive control: None mentioned
Negative control: Isotype-matched IgG

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