Easyscan pro 6

After fixation in 4% paraformaldehyde for 24–48 h, samples were embedded in paraffin and sectioned. Hematoxylin and eosin (HE) were used to examine the morphology of the implants at each study time point. Immunohistochemical staining was performed for the lipogenic marker Perilipin-1 (Abcam ab3526, Cambridge, UK, 1:1000) and CD31 (Abcam ab28364, Cambridge, UK, 1:4000) was used to locate vascular endothelial cells. Sections were scanned using a microscopic digital slice scanning system (Motic EasyScan Pro 6). Image J software was used to calculate the area proportion of panoramic Perilipin-1-positive adipocytes to assess adipogenesis in implants. The CD31-positive area proportion of 5 random fields of view (×100 magnification) was calculated to assess angiogenesis in the implants in each group.

Tissue samples (BAT, iWAT, and eWAT) were fixed in 10% formalin solution (Sigma-Aldrich, #HT501320) for 24–36 h at 4 °C, embedded in paraffin, cut at 5 µm (BAT) and 6 µm (eWAT and iWAT) thick sections, and then stained with hematoxylin and eosin (H&E). For immunohistochemistry, deparaffinization and dehydration of tissue paraffin sections were conducted with xylene and ethanol. Heat-induced antigen retrieval was performed by boiling a section in 10 mM citric acid buffer (pH 6.0) for 15 min at 95–100 °C. Slides were incubated with 3% hydrogen peroxidase to block the endogenous peroxidase activity. After washing with TBS-T buffer, sections were blocked with goat serum (1.5% blocking solution) for 1 h at room temperature followed by incubation with anti-eIF2α^Ser51 (Cell Signaling Technology, #3398) antibody diluted 1:150 or anti-UCP1 (Abcam, #ab10983) antibody diluted 1:500 at 4 °C overnight. These slides were incubated with biotinylated goat anti-rabbit IgG secondary antibody (Vector Laboratories, #PK-6101) diluted 1 drop (50 μl) to 10 ml blocking solution at room temperature for 1 h. Labeling was then visualized with 3,3’-diaminobenzidine (DAB). Nuclei were stained with hematoxylin. Slides were scanned with a MoticEasyScan Pro 6 (Motic) scanner and scanned images were viewed with Aperio ImageScope software v.12.4.3.5008 (Leica Biosystems).

After the specimens were fixed in 4 % paraformaldehyde for 24–48 h, the tissues were cut at the center of the longest diameter, embedded in paraffin and sectioned. HE was used to evaluate the morphology of the implants at each time point. The lipogenic marker perilipin-1 (Abcam, Cambridge, UK) was stained by immunohistochemistry. CD31 (Abcam, Cambridge, UK) was used to localize vascular endothelial cells. Panoramic scanning of sections was performed with Microdigital section scanning system (Motic EasyScan Pro 6). Image J software was used to calculate the area percentage of panoramic perilipin-1-positive adipocytes to assess in-implant adipocyte regeneration. Adipocytes were randomly selected to measure cell diameter (the longest diameter was calculated for non-circular adipocytes). The area percentage of CD31-positive vessels at 100x was calculated to assess in-implant angiogenesis.

For histology of AFL channels, we treated healthy skin with CO₂-laser and isolated skin biopsies shortly after. For histology of microscopic BCCs, we isolated samples from non-treated tumor-induced skin. All skin biopsies were put into paraformaldehyde-soaked nylon filters (Leica Biosystems, product number 3801085, Maarn, the Netherlands) to reduce tissue stretching, placed in tissue cassettes, and submerged in stabilized and buffered 4% formaldehyde (VWR Chemicals, catalog number 9713.1000, Leuven, Belgium). Samples were then embedded in paraffin in a Shandon Excelsior ES (Thermo Fisher Scientific, Cheshire, UK) before being cut into 3-µm-thick sections with a RM2255 microtome (RRID:SCR_020229, Leica Biosystems, Nussloch, Germany). Tissue sections were stained with hematoxylin (Merck, mixed by Apoteket RegionH, Copenhagen, Denmark) and eosin (Acros, mixed by Apoteket, Copenhagen, Denmark), and microscopy and digitalization were performed on a Motic EasyScan Pro 6 (Motic, Barcelona, Spain) using a 20× objective.