Ab48508

Fresh skin tissues of ALC and MF were fixed with 4% formaldehyde and then embedded in paraffin. They were then cut fully, and immunohistochemical was performed using 5 μm-thick sections. Skin samples were stained with the immunoperoxidase affinity biotin method using an anti-8-hydroxy-2′-deoxyguanosine antibody (N45.1, ab48508, Abcam, USA, 1:50) overnight at 4 °C followed by an HRP-conjugated Goat anti-mouse IgG as a secondary antibody for 1.5 h. The antigen–antibody reaction was observed using 3,3-diaminobenzidine tetrahydrochloride (DAB) by incubating for 5 min at room temperature. Next, an alcohol gradient was used for dehydration, and neutral gum was used to seal the slices. Images were taken under a light microscope (ECHO, RVL-100-G, USA).

We followed our previously described protocol [31 (link)]. Briefly, frozen tissue samples were homogenised in ice-cold lysis buffer and protein concentrations determined using a BCA protein assay kit (Sigma, Poole, UK). Lysates were mixed with 3 x SDS PAGE sample buffer, boiled for 5 min and allowed to cool to room temperature. Equal amounts of protein (30–50 μg) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, using 7.5–12.5% polyacrylamide resolving gels, and transferred onto nitrocellulose membrane (Invitrogen, Paisley, UK), and subjected to immunoblot analysis. Membranes were blocked for 1 h at 25 °C in 5% milk diluted in Tris-buffered saline (TBS) and 0.1% Tween 20 and incubated with the following primary antibodies overnight at 4 °C: anti-p21 (Cell Signaling; #2947), anti-p16 (Abcam; ab51243), or anti-cGAMP (Abcam; ab48508). After washing and incubating with secondary antibodies, immunoreactive proteins were visualized by the ECL plus chemiluminescence system following the manufacturer's instructions (Amersham Biosciences, Bucks., UK). Protein bands were quantified using Image J software (National Institutes of Health, http://rsb.info.nih.gov/ij/). Protein loading was normalized against Poncaeu S staining [35 (link)]. The values are expressed as a percentage of the control lysate (100%) for each experiment.

IHC was performed as in a previous study (Yukata et al., 2018 (link)). Briefly, sections were treated with sodium citrate (10 mM, 100°C) (for antigen retrieval) and H₂O₂ (10% in PBS) (for endogenous peroxidase inactivation). Next, the slices were blocked with 10% goat serum, and incubated overnight at 4°C using primary antibodies against β-galactosidase (ab203749, Abcam, Cambridge, UK), 8-hydroxy-2 deoxyguanosine (8-OHdG) (ab48508, Abcam, Cambridge, UK), Ki67 (ab15580, Abcam, Cambridge, UK), and PCNA (ab92552, Abcam, Cambridge, UK). Then, biotinylated goat anti-mouse or anti‐rabbit IgG (Sigma, Ohio, USA) were used to treat the slices, before they were incubated with Vectastain Elite ABC reagent (Fisher Scientific, Hampton, New Hampshire, USA) for 30 min. 3,3‐diaminobenzidine was used for staining, followed by counterstaining with hematoxylin. 8-OHdG, Ki67, and PCNA are mostly expressed in the nucleus; p16 and β-galactosidase are expressed in both the nucleus and the cytoplasm. The positive cell rate for 8-OHdG, Ki67 and PCNA is the ratio of the number of positive nuclei to the number of all hematoxylin-labeled cells. The positive cell rate for p16 and β-galactosidase is the ratio of the number of positive nuclei or/and cytoplasm to the number of all hematoxylin-labeled cells.