Horseradish peroxidase goat anti rabbit secondary antibody

The APP/PS1 Tg mice were anesthetized with 7% chloral hydrate, and transcardially perfused with phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in PBS. After perfusion fixation, the mouse brains were removed and fixed overnight in 4% paraformaldehyde at 4°C. Then the brains were dehydrated with 30% sucrose in PBS solution. Coronal slices (8 μm) containing both the neocortex and hippocampus were cut on a CM 1950 (Leica) freezing microtome. The brain slices were co-incubated without (blank control) or with Au₂₃(CR)₁₄ for 48 h. Then the slices were incubated with the primary mouse anti-Aβ antibody (Abcam, 1:100) followed by a horseradish peroxidase Goat Anti-Rabbit secondary antibody (Abcam). After washing, the slices were stained by using diaminobenzidine and counter-stained by hematoxylin, and observed by using an BX53 biological microscope (Olympus).

Immunohistochemical staining of CREB3L1 protein was performed on formalin-fixed paraffin-embedded human breast tumor samples obtained from the Manitoba Tumor Bank, according to the manufacturer’s instructions using Pierce Peroxidase Detection Kit (Thermo Scientific, Burlington, ON, Canada). Briefly, sections were de-paraffinized and heated in citrate buffer (pH = 6), followed by an overnight incubation at 4 °C with a rabbit polyclonal anti-CREB3L1 antibody (1:100; Protein Tech, Rosemont, IL, USA 11235-2-AP). Subsequently, slides were incubated with a horseradish peroxidase goat anti-rabbit secondary antibody (1:1000; Abcam, Toronto, ON, Canada ab6721) for 2 hours at room temperature and reacted with 3,3′-Diaminobenzidine (DAB) for 15 minutes. Sections were counterstained with hematoxylin. CREB3L1 staining was evaluated by a pathologist using two criteria, staining intensity (absent = 0, weak = 1, moderate = 2, strong = 3, very strong = 4) and % cells staining positive (0–5 % = 0, 6–49 % = 1, 50–69 % = 2, 70–89 % = 3, 90–100 % = 4). Scores were added together and described as little or no CREB3L1 staining (combined score 0–1), low (scores 2–3), medium (scores 4–5) and high (scores 6–8) CREB3L1 expression, analogous to the Allred system [39 (link)]. In addition, the subcellular location of CREB3L1 was evaluated as nuclear or cytoplasmic.