Hc pl apo 63 1.20 w corr cs2

SEM imaging was performed by Hanaichi Ultrastructure Research Institute, Co., Ltd. Collagen structure was detected by SHG imaging using an inverted two-photon microscope TCS SP8 MP (Leica) with 63× magnification with a water immersion lens (Leica, HC PL APO 63×/1,20 W CORR CS2). Fixed skin was mounted in Fluoromount and photographed under the microscope from the dermal side. Using InSight DeepSee femtosecond infra-red laser (Spectra-Physics), 900 nm was chosen as excitation wavelength, and SHG signals and GFP signals amplified with Alexa Fluor 488 were detected through filters of 435–485 nm and 500 to 550 nm, respectively. The z-step size was set to 0.8 μm.

GFP-Col1a2 and mCherry plasmid vectors were co-transfected into intact skin of axolotl limbs. The day after transfection, the in vivo GFP and mCherry signals were observed using an inverted two-photon microscope TCS SP8 MP (Leica) with 63× magnification with a water immersion lens (Leica, HC PL APO 63×/1.20 W CORR CS2). Using InSight DeepSee femtosecond infra-red laser (Spectra-Physics), 900 nm was chosen as excitation wavelength, and GFP and mCherry signals were detected through filters of 500–550 nm and 580.5 to 653.5 nm, respectively. The z-step size was set to 1 μm. Amira software was used to align the images. To visualize the structure of an entire single cell, maximum intensity projection was applied to the confocal images using Fiji software. Adjustments of brightness and contrast were applied to figure images using Fiji software.