Figure S2.
Re-photobleaching procedures for the measurement of basement membrane expansion. (A) Images of re-photobleaching procedures (see Materials and methods for detailed procedures and explanations). A z-plane exhibiting sharp and narrow signals from the BM within the targeted area was selected for photobleaching (panels a, b, and b′). The ROIs (indicated by rectangular boxes) were set on the targeted z-plane. The ROI was photobleached across 10 ± 3 continuous z-planes (with 1 µm intervals), centered on the target z-plane. For example, the BMs in the tip and lower stalk regions presented sharp and narrow signals; thus, the purple, green, and yellow ROIs were set around the main z-plane (panel b). As the upper stalk BM showed sharper signals in the 1 µm lower z-plane (additional z-plane: panel b′), the cyan ROI was set around the additional z-plane. Additional z-planes are displayed only if some target areas in the main z-plane do not show sharp eGFP-COL4A2 signals. The same photobleaching procedure was applied to each ROI. At intervals of 2–2 h and 30 min of culture following each photobleaching session, the edges of the photobleached areas were identified (distinguishable edges are indicated by arrowheads: panels c, d, f, g, i, j, and j′), and a new ROI was drawn along the identified photobleached edges (panels e, e′, h, and h′). This procedure was repeated twice until the end of the imaging session (7 h after initial photobleaching: panels j and j′). Scale bar: 20 µm. (B) Images of BM length changes. These images show the procedures used to quantify BM length changes in A. Measurements were conducted in different z-planes to determine the length of the BM at the upper and lower stalk regions (upper panels) and the tip region (upper and lower panels). Scale bar: 20 µm. (C) Maximum projection images of eGFP-COL4A2 in hair follicles in A are presented. Side views of the dotted cuboidal areas are shown. As no significant shape changes were observed at the boundaries of the 3D cuboidal bleached area during the imaging period, we can conclude that the BM maintains a consistent shape and expansion rate, even with shifts in the circumferential location of the hair follicle. This suggests that any potential displacement around the circumference does not significantly affect the accuracy of the measurements. Scale bars: 20 µm for maximum projection images; 5 µm for magnified side view images.

Re-photobleaching procedures for the measurement of basement membrane expansion. (A) Images of re-photobleaching procedures (see Materials and methods for detailed procedures and explanations). A z-plane exhibiting sharp and narrow signals from the BM within the targeted area was selected for photobleaching (panels a, b, and b′). The ROIs (indicated by rectangular boxes) were set on the targeted z-plane. The ROI was photobleached across 10 ± 3 continuous z-planes (with 1 µm intervals), centered on the target z-plane. For example, the BMs in the tip and lower stalk regions presented sharp and narrow signals; thus, the purple, green, and yellow ROIs were set around the main z-plane (panel b). As the upper stalk BM showed sharper signals in the 1 µm lower z-plane (additional z-plane: panel b′), the cyan ROI was set around the additional z-plane. Additional z-planes are displayed only if some target areas in the main z-plane do not show sharp eGFP-COL4A2 signals. The same photobleaching procedure was applied to each ROI. At intervals of 2–2 h and 30 min of culture following each photobleaching session, the edges of the photobleached areas were identified (distinguishable edges are indicated by arrowheads: panels c, d, f, g, i, j, and j′), and a new ROI was drawn along the identified photobleached edges (panels e, e′, h, and h′). This procedure was repeated twice until the end of the imaging session (7 h after initial photobleaching: panels j and j′). Scale bar: 20 µm. (B) Images of BM length changes. These images show the procedures used to quantify BM length changes in A. Measurements were conducted in different z-planes to determine the length of the BM at the upper and lower stalk regions (upper panels) and the tip region (upper and lower panels). Scale bar: 20 µm. (C) Maximum projection images of eGFP-COL4A2 in hair follicles in A are presented. Side views of the dotted cuboidal areas are shown. As no significant shape changes were observed at the boundaries of the 3D cuboidal bleached area during the imaging period, we can conclude that the BM maintains a consistent shape and expansion rate, even with shifts in the circumferential location of the hair follicle. This suggests that any potential displacement around the circumference does not significantly affect the accuracy of the measurements. Scale bars: 20 µm for maximum projection images; 5 µm for magnified side view images.

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