Figure 6. A subset of newly generated podocytes from PEC origin (tdTomato+EGFP+) co-express nephrin in the glomerular tuft of aged mice. (A–D) Representative confocal images of tdTomato staining (red fluorescent protein, RFP) (red), EGFP+ (green) and nephrin staining (blue) in young and aged mice. The inserts show separate channels of the outlined glomeruli, with superscripts: 1=tdTomato, 2=EGFP, 3=nephrin and 4=merged. (A) Young mice (OC) showed that tdTomato+PECs (red) were observed along Bowman’s capsule (A1) and genetically labeled podocytes (EGFP+ cells, green) (A2) co-express nephrin (blue) (A3), creating a cyan color (A4). (B) Aged mice (OC) showed that a subset of tdTomato+ PECs (labeled with red fluorescent anti-RFP) (B1) co-expresses EGFP+ (green) (B2) and nephrin (blue) (B3) creating a pink/yellow color in glomerular tuft (marked with white arrow) (B4). (C) Young mice (JMC) showed that tdTomato+ staining PECs were observed along Bowman’s capsule (C1) EGFP staining (C2) was detected in a typical podocyte distribution and overlaps with nephrin staining (C3), creating a cyan color (C4). (D) Aged mice (JMC) showed that tdTomato+ staining PECs (D1) (marked with white arrows) have migrated onto the glomerular tuft and differentiated to a podocyte fate, co-staining with EGFP (green) (D2) and nephrin (blue) (D3) creating pink/yellow color (D4). Scale bars represent 25μm or 5μm (insets).