Advanced image analysis with ImageJ/Fiji software

False nuclear LDLR staining was removed by separating the channels of the immunofluorescence images. The nuclei channel was analyzed with “threshold”, “count particle”, “create mask”, “select all nuclei”, and “add to ROI manager”. Then, the channel for LDLR staining was selected, “image overlay from ROI”, “delete”, “no selection”, “convert to gray image”, “set threshold”, “analyze particles”: 5 for 60× and 12 for 40×‐Infinity (μm²). Then, area (cluster size) was measured and mean area to number of nuclei was calculated.

Channels of the immunofluorescence images were separated. Podocalyxin (vessel marker) channel was selected, threshold was set and detected area was converted to mask and added to ROI manager. Then, the filipin channel was selected and overlaid from ROI. Signal in ROI area was measured. Mean integrated density was determined for 10 images per animal/condition and normalized to wild‐type mice.