Immunohistochemistry

Immunohistochemistry for p16, p21, γH2AX and 8‐OHdG (8‐hydroxydeoxyguanosine) was performed on FFPE tissue using a standard avidin‐biotin horseradish peroxidase enzyme complex method (Vectastain Universal Elite ABC kit, Vector Laboratories, UK), and the signal was visualized using 3,3′‐diaminobenzidine (DAB) (Vector Laboratories, UK). Briefly, 6‐μm sections were deparaffinized and rehydrated to water. Endogenous peroxidase activity was quenched by incubation of the sections in 0.3% H₂O₂/methanol for 20 min at room temperature (RT). After antigen retrieval, sections were incubated in 1.5% normal serum for 30 min at RT, followed by incubation with the respective primary antibody. A summary of the primary antibodies, all commercially available and optimized for this study, and their conditions of use is shown in Table 2. Sections were then incubated with a biotinylated secondary antibody (against specific species depending on primary antibody used) for 30 min at RT, followed by incubation with ABC reagent, for 30 min at RT. To visualize the signal, sections were incubated with the peroxidase substrate solution, then counterstained with haematoxylin, cleared and mounted. Negative controls consisted of sections incubated with omission of the primary antibody and isotype controls.

Source, specificity, dilution, antigen retrieval and incubation conditions for the antibodies used for immunohistochemistry and double immunofluorescence.

EDTA, ethylenediaminetetraacetic acid buffer; MW, microwave; NA, not applicable; O/N, overnight; RT, room temperature; TSC, trisodium citrate buffer; 8‐OHdG, 8‐hydroxy‐2′‐deoxyguanosine.

To determine whether glial cells expressing p16 and p21 were astrocytes, double immunofluorescence against p16 or p21 and glial fibrillary acidic protein (GFAP) was performed. For this, 8‐μm frozen sections were fixed in acetone and incubated with 0.2% glycine. Sections were blocked with normal serum (1.5%) for 30 min at RT, followed by an overnight incubation at 4°C with rabbit anti‐GFAP and mouse anti‐p21 or mouse anti‐p16. Sections were then washed and incubated with Alexa Fluor fluorescent secondary antibodies (Thermo Fisher, Waltham, MA, USA) goat anti‐mouse (Alexa Fluor 568, 1:500) and donkey anti‐rabbit (Alexa fluor 488, 1:500) for 1 h at RT. Sections were incubated with Hoechst 33342 solution (Sigma‐Aldrich, St. Louis, MO, USA) to stain the nuclei before mounting with Fluoromount Mounting Media (Sigma‐Aldrich, St. Louis, MO, USA). Double immunofluorescent staining was also performed to confirm the expression of p21 in neurones. For this, acetone‐fixed frozen sections were incubated with rabbit anti‐NeuN (which recognizes nuclear and cytoplasmic NeuN isoforms) and mouse anti‐p21 or mouse anti‐p16 following the protocol described previously. Every run included single‐labelled sections that showed the same staining pattern as seen in the double‐labelled sections. Details of the antibodies used for immunofluorescence experiments are specified in Table 2. Images were captured with a Nikon Eclipse 80i microscope (Nikon UK, Kingston upon Thames, UK), and co‐localization was assessed using the Fiji ImageJ image processing package 24.