Ab178847

Animals were deeply anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and underwent sternotomy, followed by intracardiac perfusion with 200 ml saline and 200 ml 4% ice-cold paraformaldehyde in 0.1 M phosphate-buffered saline. The brain was removed, postfixed in 4% paraformaldehyde for 4 h, and subsequently allowed to equilibrate in 30% sucrose in phosphate-buffered saline overnight at 4°C. Brain samples were embedded in Tissue-Tek O.C.T., cut at a thickness of 20 μM using a cryostat (Leica CM1850-1-1), and mounted on slides. All Brain samples were thoroughly rinsed in PBS to remove any residual OCT. The samples were then blocked in 0.3% Triton-X 100 and 5% normal donkey serum (NDS) in PBS for 1 h, and incubated overnight with anti-MHCII (1:50, MCA46GA, BIO-RAD), anti-IBA-1 (1:100, ab178847, Abcam), anti-GFAP (1:100, ab254082, Abcam), anti-NEUN (1:100, ab177487, Abcam). On the next day, the samples were rinsed in PBS and incubated for 1 h in PBS containing appropriate secondary antibody (1:500) from Jackson ImmunoResearch (catalog nos. 711-545-152 or 715-585-150) or Cell Signaling Technology (catalog nos. 8,889 or 4,408). The samples were then rinsed in PBS and coverslipped with Antifade Mounting Medium with DAPI (Beyotime, Nanjing, China). The imaging and subsequent analysis were performed using a Confocal Microscope (TCS SP8, Leica Microsystems, Mannheim, Germany).

Brains were snap frozen in 2-methylbutane cooled in liquid nitrogen and embedded in OCT before cutting. Coronal sections (10 μm) were cut using a freezing sliding microtome (Leica CM1850) and stored at –20°C until use. For S100β and IBA1 immunostaining, slides were washed two times with PBS, blocked and permeabilized with PBS 1% bovine serum albumin, 5% goat serum (Biological Industries, Israel) and 0.05% Triton-X (Sigma-Aldrich) for 1 h and incubated with S100β antibody (1:500, ab66028, Abcam) or IBA1 antibody (1:200, ab178847, Abcam) overnight in 4°C. Slides were washed 3 times with PBS and incubated with secondary goat anti mouse antibody (1/700, Alexa-Flour) for 1 h at room temperature. DNA was stained with DAPI (1:1000, Sigma-Aldrich). For Thioflavin S (ThioS) staining, slides were incubated for 8 min with 0.01% ThioS solution in 50% ethanol. Slices were then briefly incubated twice for 10 s with 80% ethanol, and washed twice with double distilled water (DDW).

The cerebral cortex was fixed in 10% formalin, embedded in paraffin, and cut into 3 μm sections. After dewaxing, dehydration and antigen retrieval, the sections were incubated with 3% hydrogen peroxide solution at room temperature for 25 min to quench endogenous peroxidase. After blocked by 3% BSA, the sections were incubated with anti-Iba1 antibody (1:8000, ab178847, Abcam) at 4 °C for 24 h. Then, the sections were incubated at room temperature for 45 min with secondary antibodies labeled with horseradish peroxidase (HRP). Afterwards, the slides were incubated in the chromogen 3,3′ -diaminobenzidine (DAB; Vector) at room temperature and coloration time was controlled through a microscope. Sections were then washed in running tap water, counterstained with hematoxylin, and mounted. Images were taken using an inverted microscope (Nikon, Japan).

One set of serial sections containing the hippocampus was randomly chosen from each group of rats. Free-floating sections were rinsed with PBS + T 6 times for 10 min each. Then the sections were blocked with 1% FBS, 10% SP 9001-A, and PBS + T for 2 h at 37 °C. The sections were then incubated with a rabbit anti-Iba1 primary antibody (1:500, ab178847, Abcam, Cambridge, UK) and a mouse anti-CD68 primary antibody (1:100, ab955, Abcam, Cambridge, UK) in PBS + T for 2 days at 4 °C. The sections were washed three times for 10 min at room temperature in PBS + T. For the immunofluorescence staining, the sections were incubated with DyLight 549- and DyLight 488-labeled secondary antibodies (1:200; Abbkine, USA) for 2 h at 37 °C. 4’,6-Diamidino-2-phenylindole (AR1176, Boster, Wuhan, P. R. China) was used for the nuclear staining. Fluorescent images were captured at a ×200 magnification under a Zeiss fluorescence microscope (Zeiss, Germany). At least ten representative images of each hippocampal subregion were acquired from each rat. The Iba1⁺/CD68⁺ cells in each image were manually quantified, and the cell density and percentage were calculated and analyzed. The percentage of activated microglia is expressed as the ratio of Iba1⁺/CD68⁺ cells to Iba1⁺ cells.

Dilutions of C. pneumoniae bacteria were prepared in Dulbecco’s phosphate buffered saline (DPBS). OECs, TgSCs, astrocytes and microglia were seeded at the density of 4000 cells/well in 96-well plate (Costar) in glial medium. After 24 h, bacteria (multiplicity of infection (MOI): 1:1) were added and incubated with cells for 72 h. Following the infection, the cells were then rinsed in 1 × DPBS and were fixed for 20 min in 4% PFA in DPBS. Subsequently, cells were washed and incubated in blocking buffer for 1 h. Cells were then incubated with the following primary antibodies overnight at 4 °C; goat anti-C. pneumoniae/Chlamydia trachomatis (Abcam, ab20929; 1:400) and rabbit anti-glial fibrillary acidic protein (GFAP) antibody (Thermofisher Scientific, PA5-16291; 1:200) or rabbit anti-ionized calcium-binding adaptor molecule 1 (IBA1) microglia (Abcam, ab178847; 1:100). The following day, cells were washed with DPBS and incubated with secondary antibody donkey anti-goat Alexa Fluor 488 (Thermofisher Scientific, A11055; 1:400) and goat anti-rabbit 647 (ThermoFisher Scientific, A32733; 1:400) for 1 h. Nuclei were stained with DAPI. Hep-2 cells were visualized by CellMask Orange Plasma membrane stain (Thermofisher Scientific, C100455; 1:10,000).