Abc system

For PARP-1, immunohistochemical (IHC) staining of FFPE brain tissue was performed as previously described [22 (link)]. Briefly, the samples were de-paraffinized with xylene 3x 3 min, before being rehydrated in 100% anhydrous alcohol 2x 3 min followed by 95%, 70%, and 50% ethanol for 3 min each then rinsed in running tap water and distilled water. After rehydration, antigen retrieval was undertaken with 10 mM citrate buffer (pH 6.0) while being microwave irradiated for 15 min, followed by incubation with primary antibody (PARP-1 monoclonal antibody, 1:200; Cat # 1522G, AbD Serotec), then with biotinylated secondary antibody horse anti-mouse (1:200, Vector Laboratories), and developed using the ABC system (Vector Laboratories). H&E staining was performed as per standard protocol, briefly as follows: Sections were de-paraffinized with xylene 3x 3 min, before being rehydrated in 100% anhydrous alcohol 2x 3 min followed by 95%, 70%, and 50% ethanol for 3 min each. The samples were briefly rinsed in running tap water then distilled water. Staining was done with Richard-Allan Hematoxylin for 30s followed by a tap water/clarifier rinse for 30s, tap water rinse for 30s, bluing reagent for 1 min, another tap water rinse for 1 min, a rinse in 95% dehydrant alcohol followed by Richard-Allan Eosin-Y for 90s, 100% anhydrous alcohol for 3 min, and finally xylene for 3 min [31 (link)].

The postmortem tissue of the AD cases was provided by BRAIN UK under the ethics approval obtained from the National Research Ethics Committee South Central Hampshire B (REC reference 14/SC/0098). Formalin-fixed paraffin-embedded brain tissue was cut into 5 µm sections on a microtome. Tissue was dewaxed in clearene (Surgipath), and rehydrated in graded alcohols. Endogenous peroxidase activity was quenched by incubation of tissue with 3 % H₂O₂ in methanol for 10 min, before 3 washes with tris-buffered saline (TBS). Sections were then either incubated for 30 min in 80 % formic acid (Fisher) or in TBS. Sections were blocked for 30 min with medium containing DMEM, FCS BSA. Anti-Aβ antibodies were added at a concentration of 1:1000 in TBS and incubated at 4 °C overnight. Sections were then washed in 3 rinses of TBS before addition of biotinylated rabbit anti-mouse IgG secondary (Abcam, 1:600) and incubated for 1 h. Bound antibody was detected by incubation for 30 min with ABC system (Vector), and washed 3 times in TBS, before developing with diaminobenzidine solution (DAB, Vector). Slides were dehydrated in graded alcohols into clearene, before mounting with pertex™ (Cellpath).

The brain sections were retrieved in citrate buffer (pH 6.0) at 95°C for 30 min, then incubated in 2 N HCl for 30 min at 37°C and 0.1 M borate buffer (pH 8.5) for 15 min. After washing in 0.01 M PBS, the sections were incubated overnight with the anti-BrdU (1:1000, Abcam, United Kingdom), and then incubated with the biotinylated goat anti-rat IgG (1:200, Dako, Denmark). The BrdU staining was visualized with the peroxidase method (ABC system, Vector Laboratories, Burlingame, CA, United States) and diaminobenzidine kits (DAB kits, Sigma-Aldrich, United States). For doublecortin (DCX) or Ki67 staining, sections were incubated with rabbit anti-DCX (1:200; Abcam, United Kingdom) or rabbit anti-Ki67 (1:1000; Novocastra) antibody, followed by the biotinylated goat anti-rabbit (1:200; Dako, Denmark) and visualized using the same methods mentioned above.
Immunofluorescent colabeling of BrdU and DCX was performed. After antigen retrieval, sections were incubated with primary antibodies overnight and secondary antibodies for 2 h, including goat anti-rabbit IgG Alexa Fluor-488 and goat anti-rat IgG Alexa Fluor-568 (1:200, Dako, Denmark). The mounted sections were observed by fluorescent microscopy (Axioplan, Zeiss, Oberkochen, Germany).