RNA Fluorescence in situ hybridization (RNA FISH)

FFPE blocks of young (28–37 y/o) and old (54–89 y/o) skin donors, fixed in 4–10% formalin and cut into 4 µm sections, were subjected to RNA FISH using the RNAScope Multiplex Fluorescent Detection Kit v2 (ACDBio, cat. no. 323100) following manufacturer’s instructions with extended pretreatment of the samples. These pretreatment steps included a 15 min incubation with hydrogen peroxide, a mild boil (98–102 °C) for 30 min with target retrieval reagents and a 30 min incubation at 40 °C in the HybEZ^TM (ACDBio) oven with Protease Plus (all solutions as included in the kit). Probes against human CTHRC1 (ACDBio, cat. no. 413331), APCDD1 (ACDBio, cat. no. 535851-C2), CCL19 (ACDBio, cat. no. 474361), APOE (ACDBio, cat. no. 433091-C2), ASPN (ACDBio, cat. no. 404481), CD248 (ACDBio, cat. no. 542501), and PDGFRA (ACDBio, cat. no. 604481-C3) mRNA molecules were used. Nuclei were counterstained with DAPI and mounted using ProLong Gold Antifade Mountant (ThermoFisher, cat. no. {"type":"entrez-protein","attrs":{"text":"P36930","term_id":"1248281091","term_text":"P36930"}}P36930). We performed the experiment in three young and three old sections for each target gene, always including PDGFRA as a pan-fibroblast marker. Images were taken with a TCS SP5 confocal microscope (Leica Microsystems) using 40X oil immersion lens and were further processed using the Fiji software⁶⁶.

APCDD1- and CTHRC1-positive cells were quantified in two images per dermal region (papillary dermis, reticular dermis and deep reticular dermis) for each skin section in young and old samples. Statistical analysis of the quantification of APCDD1- and CTHRC1-positive cells was performed using a two-way ANOVA test with Dunnett correction, which compared the percentage of positive cells in the reticular and deep reticular areas to the percentage present in the papillary area for each gene independently.

CD248 positive cells were quantified in two images of deep reticular dermis for each skin section in young and old samples, respectively. Statistical analysis was performed using an unpaired two-sided t-test, comparing the percentage of CD248 positive cells.