Histoclear

Samples were fixed in 4% paraformaldehyde and dehydrated through ethanols. Samples were incubated in Histoclear (Scientific Laboratory Supplies, Nottingham, UK) alone, with molten paraffin wax (ThermoFisher Scientific) and paraffin wax alone. Samples were embedded in plastic molds (CellPath, Newton, UK) with paraffin wax and sectioned transversely using a microtome (Leica RM2125RT). 5 μm sections were placed onto charged microscope slides (ThermoFisher Scientific).

Sections were deparaffinised in Histo‐Clear and sequentially rehydrated from 100% ethanol to distilled water, before incubating in Mayer's haematoxylin (Sigma‐Aldrich) for 5 min. Sections were washed in distilled water, submerged in alkaline ethanol for 30 s and sequentially dehydrated to 95% ethanol. Samples were counter‐stained in eosin (Sigma‐Aldrich) for 30 s and then dehydrated to 100% ethanol. Sections were incubated twice in Histo‐Clear then mounted in Omnimount (Scientific Laboratory Supplies) prior to imaging. Samples were imaged using a Leica microscope, and images were captured using the Leica EZ software. Four sections per skin biopsy (n = 40 skin biopsies, n = 160 sections) were stained and imaged along their entire length at 20× magnification, and images were stitched together using Fiji software to visualise the complete skin section.
¹⁸ (link) These images were used for quantification of epidermal thickness, interdigitation index, rête ridge morphology and dermal papilla morphology (Figure S1A,D–F).

Skin biopsies were fixed in 4% paraformaldehyde (Sigma‐Aldrich), serially dehydrated through a series of ethanol solutions (30%–100% v/v), then incubated in Histo‐Clear (Scientific Laboratory Supplies), and a 1:1 ratio of Histo‐Clear and paraffin wax (Thermo Fisher Scientific). Models were further incubated in paraffin wax prior to embedding in plastic moulds (Solmedia Ltd). Paraffin wax blocks were sectioned transversely at 5 μm using a microtome (Leica) and transferred onto charged microscope slides (Thermo Fisher Scientific) for analysis.

Immunofluorescence was carried out on paraffin embedded organotypic raft culture sections using the agitated low temperature epitope retrieval (ALTER) method as previously described [39 ]. Briefly, slides were sequentially immersed in Histoclear (Scientific Laboratory Supplies) and 100% IMS and incubated at 65°C in 1 mM EDTA (pH 8.0), 0.1% Tween 20 overnight with agitation. Slides were then blocked in PBS containing 20% heat-inactivated normal goat serum and 0.1% BSA (Merck). Primary antibodies were diluted in block solution and incubated overnight at 4°C followed by 3x PBS washes. Fluorophore-conjugated secondary antibodies were diluted in block buffer and added to slides which were incubated at 37°C for 1 hour. Slides were subsequently washed 4x 10 mins in PBS with Hoechst 33342 solution (10 μg/ml) added to the final PBS wash. Slides were mounted in Fluoroshield (Sigma-Aldrich) and visualised using a Nikon inverted Epifluorescent microscope fitted with a 40x oil objective. Images were captured using a Leica DC200 camera and software.

Skin equivalents and human skin samples were fixed in 4% paraformaldehyde and gradually dehydrated in ethanol and incubated in Histoclear (Scientific Laboratory Supplies, Nottingham, United Kingdom), Histoclear:paraffin wax and 100% paraffin wax (CellPath, Newton, United Kingdom). Samples were embedded in plastic moulds (Solmedia, Shrewsbury, United Kingdom) with paraffin wax. Paraffin wax blocks were sectioned at 5 μm using a rotary manual microtome Leica RM2125RT (Leica Biosystems, Nussloch, Germany) with MB DynaSharp microtome blades (Thermo Fisher Scientific). Transverse sections were mounted onto charged Superfrost Plus microscope slides (Thermo Fisher Scientific).

Before immunofluorescence staining, cryosections were hydrated in PBS at room temperature (RT) for 10 min, and paraffin‐embedded sections were deparaffinised and hydrated as follows: histoclear (Scientific Laboratory Supplies, Wilford) 2× for 5 min, followed by ethanol 100% 2× for 5 min, ethanol 90% for 5 min, ethanol 70% for 5 min and distilled water 2× for 5 min. After antigen retrieval in 0.01 M citrate buffer at pH 6.0 for 10 min, the sections were permeabilised in PBS 0.5% Triton X‐100 for 10 min and blocked in 3% bovine serum albumin (BSA), 5% Goat Serum (or Donkey Serum), 0.3% Tween‐20 in PBS, for 1 h. The slides were then incubated with the primary antibody at 4°C ON. After washes in PBS 0.1% Tween‐20 (3× 10 min) to remove excess to primary antibody, the sections were incubated with secondary antibody at RT for 1 h. Finally, the slides were incubated in 1 μg/ml of 4′,6‐diamidino‐2‐phenylindole (DAPI, Thermo Fisher Scientific) at RT for 10 min, washed in PBS 1×, and mounted with vectashield (Vector Laboratories). The primary and secondary antibodies used in this study are described in Tables 1 and 2 respectively.

Samples were fixed in 4% paraformaldehyde (Sigma‐Aldrich) overnight at 4°C and then dehydrated through a series of ethanols. Samples were incubated in Histoclear (Scientific Laboratory Supplies, Nottingham, UK) alone, then mixed 50:50 with molten paraffin wax (Thermo Fisher Scientific) followed by paraffin wax alone. Samples were embedded in plastic molds (CellPath, Newton, UK) with paraffin wax and sectioned transversely using a microtome (Leica RM2125RT). The 5 μm sections were captured onto charged microscope slides (Thermo Fisher Scientific).