Dab staining kit

Immunohistochemistry was performed to measure GRP78 and CHOP expression. The brain paraffin slices were passed through xylene and ethanol and then blocked using 0.3% hydrogen peroxide solution for 15 min. Slices were subjected to microwave antigen retrieval with citric acid buffer (PH6.0) and blocked with 5% BSA for 2 h. Then the slices incubated with GRP78 antibody (1:2000, Cell Signaling Technology, Inc., Beverly, MA, USA) and CHOP antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. Next, slices were incubated with avidin-biotin horseradish peroxidase complex (Boster Biotechnology, China) for 30 min at 37°C. To develop color, slices were incubated in a DAB staining kit (Boster Biotechnology, China) and counterstained with hematoxylin. The positive cells were stained brown under light microscope. The fields (200×) of the hippocampus CA1 region of eight sections were analyzed. The expression of GRP78 and CHOP were measured by ImageJ software by observing the density of immunopositive neurons.

The immunohistochemical method [22 (link)] was used to detect PGC-1α, Bax, cytochrome C, and Bcl-2 expression. Brain paraffin sections were dewaxed and hydrated with xylene and ethanol, and then incubated with a 0.3% hydrogen peroxide solution for 15 min. The sections were placed in citric acid buffer (pH6.0), and the antigen was repaired using a microwave. The sections were blocked with 5% BSA at 37 °C for 30 min, then the sections were incubated with antibodies for PGC-1α (1:500), Bax (1:200), cytochrome C (1:150), and Bcl-2 (1:300) at 4 °C overnight. Next, the sections were incubated with goat anti-rabbit secondary antibody (BOSTER Biological Technology Co., Ltd., Wuhan, China) at 37 °C for 30 min. Then, the sections were stained using a DAB Staining Kit (BOSTER Biological Technology Co., Ltd., Wuhan, China), and then stained with hematoxylin. The positive cells were brown under a light microscope. From each group, three sections were randomly selected and three fields from each section were selected (400×). The average optical density of positive cells was observed, and the expression of PGC-1α, Bax, cytochrome C, and Bcl-2 was analyzed by IPP software.

After dewaxing and hydration, the tissue sections were permeabilized with 0.2% triton (Sigma, St. Louis, MO, USA) at room temperature for 10 min, and then incubated with blocking solution (3.75% BSA/5% goat serum; Zymed, Carlsbad, CA, USA) for 30 min. Next, samples were incubated with anti-CXCL11 (CSB-PA06119A0Rb, Cusabio). All sections were incubated with poly-HRP antibody for 30 min. Then, sections were stained by DAB staining kit (Boster, Wuhan, China), and then stained with hematoxylin (0.2 mg/ml, Sigma) to label the nucleus. Finally, sections were observed under a microscope.