Tsc sp8

Proximal, mid and distal segments of the SI were fixed with 4% paraformaldehyde (PFA) from 3 h to overnight at 4 °C, embedded in OCT and kept at −80 °C until sectioning. Sections (7 µm thick) were prepared with a cryotome (Cryostat CM 3050S).
Images from Hematoxyline QS (Vector) stained were acquired using NDP Zoomer Digital Pathology (Hamamatsu) and subsequently analysed in NDP.view2 software.
To perform immunofluorescence, sections were blocked and permeabilized in 10% adult bovine serum (Sigma), 5% skim milk (Sigma) and 0.3% Triton X-100 in PBS (blocking buffer) for at least 1 h at 4 °C. Primary antibodies (listed in Supplementary Table 3) were incubated overnight in blocking buffer at 4 °C. Alexa-Fluor-Conjugated secondary antibodies (indicated in Supplementary Table 3) were incubated for 1–2 h at room temperature in 10% adult bovine serum and 0.1% bovine serum albumin (BSA) (Sigma) in PBS. Diamidino-2-phenylindole dihydrochloride (DAPI; 1 µM; Sigma) was used to counterstain nuclei in the indicated experiments. Images were acquired using laser-scanning confocal microscopes (Leica TSC SP8 and Zeiss LSM800). All images were subsequently analysed with Fiji software.

Whole-mount immunostaining of mouse dorsal skin was performed as follows^{15 (link)}. Briefly, mouse skin tissues were dissected and fixed with 4% paraformaldehyde (PFA)/PBS for 1 h at 4 °C, and embedded in OCT compound after washing with PBS. For acetone fixation, dissected skin was directly embedded in OCT compound. Skin sections (150 μm thick) were made using a cryostat (Leica, Wetzlar, Germany) and washed with PBS. Acetone fixation was performed by placing skin sections in −30 °C acetone for 15 min, followed by acid treatment with 0.1 N HCl/0.1 N KCl for 15 min after washing in PBS. Skin sections were blocked with a blocking buffer (0.5% skim milk/0.25% fish skin gelatine/0.5% Triton X-100/PBS) for 1 h at 4 °C, and then incubated with primary antibodies diluted in blocking buffer overnight at 4 °C. Skin samples were washed with 0.2% Tween 20/PBS for 4 h and then incubated with secondary antibodies similarly to the primary antibodies. After that, skin samples were stained with DAPI, washed with 0.2% Tween 20/PBS for 4 h at 4 °C, and mounted with BABB clearing solution. Images were acquired using Leica TSC SP8 and Zeiss LSM880 with Airyscan (for 3D movie of the hook BM). Three-dimensional reconstructed images were produced using Imaris software (Bitplane, Oxford, UK).