The skin explants were fixed in 10% neutral buffered formalin for 48 h at 4 °C, dehydrated and embedded in paraffin. Then, 5 µm-thick sections were deparaffinized in xylene and then rehydrated through a series of decreasing ratios of alcohols to water. The immunohistochemical analysis was performed as previously described [45 (link)]. The tissues were incubated with primary antibodies for 4HNE (dil. 1:400) (AB5605, Millipore Corporation, Burlington, MA, USA), type I collagen (dil. 1:200) (AB138492, Abcam, Cambridge, UK) and Filaggrin (dil. 1:50) (sc-66192, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) in 0.25% BSA in PBS overnight at 4 °C, and with fluorochrome-conjugated secondary antibodies (Alexa Fluor 568, A11004 or Alexa Fluor 488, A11055) diluted 1:1000 in 0.25% BSA in PBS at room temperature for 60 min. Nuclei were stained with DAPI (dil. 1:50,000) (D1306, Invitrogen, Waltham, MA, USA) in PBS. Coverslips were mounted onto glass slides using Fluoromount-G™ Mounting Medium (00-4958-02, ThermoFisher Scientific, Waltham, MA, USA). The tissues were examined using a Zeiss Z1 AxioObserver LSM10 confocal microscope at 40× magnification, and the images were quantified using ImageJ software 1.53a (Java 1.8.0_172, National Institutes of Health, Bethesda, MD, USA).
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