2.2. Immunofluorescent staining

Tissue samples were initially formalin-fixed and paraffin-embedded (FFPE). Subsequently, 5-micrometer-thick sections were prepared using an ultramicrotome. Prior to staining, slides were incubated overnight at 60°C to enhance tissue adhesion. The sections then underwent a deparaffinization process with xylene, followed by rehydration through an ethanol and deionized water gradient. Antigen unmasking was achieved using a 1% low pH antigen retrieval solution (Antigen Unmasking Solution, Citric Acid Based, Vector Laboratories, Inc., cat. #H-3300) heated to boiling, with slides incubated in the solution for an hour as the solution cooled to room temperature. Slides were rinsed in a 1X Phosphate Buffered Solution with 0.2% Fish Skin Gelatin (PBS-FSG) wash buffer prior to application of blocking agent for 40 min (normal goat serum, MP Biomedicals, LLC., cat. #2939149; or normal donkey serum, GeminiBio, cat. #100–151). For p16 staining, anti-p16^INK4a (Invitrogen, Ms. Mab IgG1, MA5-17093, 1:100) was applied for 1 h at room temperature. For SIRT1 staining, anti-SIRT1 (Lifespan Biosciences, Inc., Gt Pab, cat. #LS-B8356, 1:100) was applied overnight at 4°C. Secondary antibodies (Goat anti-Mouse IgG (H + L), Alexa Fluor™ 633, Invitrogen, cat. #A-21052; Donkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568, Invitrogen, cat. #A-11057) and a conjugated anti-GFAP (Anti-Glial Fibrillary Acidic Protein (GFAP) − Cy3™ antibody, Sigma-Aldrich®, Ms. Mab, cat. #C9205, 1:300; GFAP Monoclonal Antibody (GA5), Alexa Fluor™ 488, eBioscience™, Ms. Mab, cat. #53–9,892-82, 1:300) were applied for 1 h at room temperature. Slides were washed twice in 1X PBS-FSG with 0.1% Triton-100 for 5 min and once in 1X PBS-FSG for 5 min after each antibody application. Finally, slides were washed for 5 min in 1X PBS prior to adding 20 μL of EverBrite TrueBlack® Hardset Mounting Medium (Biotium, cat. #23018) and a coverslip. Slides were left to dry overnight at room temperature before being imaged.