Figure 4. High-resolution imaging to visualize collagen IV α3α4α5 network formation.

(A) High-resolution imaging of glomeruli from P3h2^ΔPod and P3h2^fl/fl mice with expansion microscopy. In WT GBM, a linear collagen IV localization was observed for collagen IV and collagen IV α3, α4, and α5 proteins, indicating proper network formation. However, in KO GBM, collagen IV alignment was split, and irregular network formation (white arrows) was observed for collagen IV, collagen IV α3, α4, and α5 stainings, which showed disrupted network formation. Microscopy was performed with a LSM 800 with Airyscan using a ×63 objective, a digital zoom of ×8, and ×4 linear expansion of tissues. (B) Quantification of irregular network formation of collagen IV. Randomly chosen glomeruli from WT and KO mice were evaluated for linear and irregular collagen IV network formation. The graph shows that collagen IV network formation in KO GBM was significantly disrupted when compared with that in WT GBM, indicating that collagen IV α3, α4, and α5 proteins were unable to form a proper network in the absence of P3H2. n = 3. Graphs show the mean ± SD. ****P < 0.0001, by unpaired, 2-tailed Student’s t test.