SOD3 and EPO mRNA distribution in kidneys of adult mice. (A) Typical autoradiograph of Northern blot analysis showing SOD3 mRNA levels in kidney. Lanes 1, 2, 3, and 4 are medulla (M), proximal tubules (PT), cortex (Co), and total kidney, respectively. β-actin was used for normalization and verification of RNA loading. (B) Localization of SOD3 mRNA transcripts in mouse kidney by in situ hybridization. Paraformaldehyde-fixed renal sections (5 μm) were prepared from normal mice and hybridized with digoxigenin-labeled SOD3 antisense. (i) Wt kidney; the majority of the positive (dark blue) in situ labeling was observed in proximal tubules (p) of the cortex and outer medulla. The glomeruli (g), distal tubules, and the blood vessels (*) exhibit no significant staining. (ii) KO kidney; note absence of significant in situ staining. Original magnification, × 220 for all sections. (C) EPO mRNA localization by in situ hybridization in mouse kidney after 6 hours of hypoxia. (i) Low-power view of in situ red fluorescence staining of EPO mRNA; most labeling was observed in proximal tubules of cortex. (ii) High-power view of fluorescence staining in tubules near glomeruli but not in the tufts. (iii) Low-power view of EPO mRNA by DAB staining reveals structural detail and stain distribution in proximal tubules and capsular epithelium (p, proximal tubule). Glomeruli (g), distal tubules, and peritubular cells exhibit no or minimal staining. (iv) High-power view of details of proximal tubule staining for EPO mRNA. Final magnifications were × 220 (Ci), × 280 (Ciii), and × 550 (Cii,Civ). Objectives: × 20 (B, Ci, Ciii), × 40 (Cii, Civ).