Dab substrate kit with nickel

Immunohistochemical analyses were performed as previously reported.⁴⁷, ⁴⁸ Mouse brain tissues were placed in 10% neutral buffered formalin overnight, followed by paraffin embedding and sectioning. Sagittal sections of the brain were stained for Aβ with the 4G8 antibody (#800701, Biolegend, CA, USA). Sections were visualized by using DAB substrate kit with nickel (Vector Laboratories, CA, USA) and mounted using DPX mounting medium (Electron Microscopy Sciences). Approximately 4–6 slices per animal were taken and analyzed in the ImageJ (NIH) software and quantified using burden threshold.

miR-33^−/− mice and their littermates were placed at 4 °C as cold exposure for 2 h. miR-33b^+/+ mice and their littermates were used at room temperature. After anesthetization, mice were perfused transcardially with ice-cold PBS (pH 7.4) followed by 4% PFA, then the brains were harvested. After overnight post-fixation with 4% PFA, brain samples were cryoprotected in a 30% sucrose solution. The brain tissues were embedded in O.C.T. Compound (Tissue-Tek, Japan) and sliced at a thickness of 40 μm. The slices were treated with methanol supplemented with 0.3% H₂O₂, blocked with 1% normal donkey serum containing 0.1% TritonX-100, and then incubated with a goat anti-c-fos primary antibody (1:200) overnight. After washing with T-PBS, slices were incubated biotin-conjugated anti-goat secondary antibody (1:500) for 1 h. After washing with T-PBS, sections were incubated with VECTASTAIN Elite ABC kit (Vector Laboratories, CA) for 30 min, then the color was developed using a DAB Substrate kit with nickel (Vector Laboratories). After washing with T-PBS, slices were embedded and coverslipped. c-fos-positive cells in the rRPa were counted. The mean number of c-fos-positive cells from six sequential sections are presented here. Data were obtained by Axio Observer 7 and Zen 2 pro software (Carl Zeiss, Inc).