Ab237968

After the behavioral tests, ten mice from each group were selected randomly and a total of twenty mice were used for immunofluorescence assay to study the cellular expression of CD68 [27 (link)]. The brain tissues were dehydrated, embedded in paraffin, and prepared in 5 μm sections. The sections were deparaffinized with xylene, rehydrated, and washed with phosphate-buffered saline (PBS) for 5 minutes three times. Antigen retrieval was achieved, using a 10 mM citrate buffer (pH = 6.0) for 15 minutes at 96°C. All sections were blocked in 2% milk for 1 hour and probed with the anti-CD68 antibodies (1 : 1000, ab237968, Abcam) overnight at 4°C. After washing, the sections were incubated for 2 hours at room temperature and exposed to anti-rat/anti-rabbit IgG secondary antibody (ZB-2301, 1 : 500, Beijing ZhongShan Inc.). Cellular CD68 expression was analyzed using a computer-assisted microscope (Neurolucid™, MicrobrightWled, USA) and an image analysis system. Quantitative analysis was conducted by randomly counting CD68-immunolabeled cells under a light microscope at 400x magnification throughout 24 nonoverlapping regions of the brain tissue (0.025 mm² each) [26 (link)]. The positive cell ratio was calculated for all representative images.

The cells growing on slides were fixed with 4% of paraformaldehyde, washed three times with PBS, and then permeated with 0.5% Triton X-100 (diluted with PBS) for 20 min at room temperature. After washing the slides three times with PBS, 1% bovine serum albumin (BSA) as a blocking agent was cultured with cells at room temperature for 30 min. Then, absorbing the sealed solution with absorbent paper, a sufficient amount of anti-CD68 (M1 marker, ab237968, Abcam), anti-iNOS (M1 marker, ab49999, Abcam), anti-CD206 (M2 marker, ab64693, Abcam), anti-arginase1 (M2 marker, ab91279, Abcam), and anti-insulin degrading enzyme (ab32216, Abcam) primary antibody, diluted with BSA (diluted 1,000 times to 1 μM), was added to each slide drop and placed in a wet box, which was incubated at 4°C overnight. Next, the goat anti-rat IgG H&L Alexa Fluor 488-conjugated (ab150157, Abcam), donkey anti-rabbit IgG H&L-conjugated (Alexa Fluor® 488) (ab150073, Abcam), goat anti-mouse IgG H&L-conjugated (Alexa Fluor 594) (ab150116, Abcam), and donkey anti-rabbit IgG H&L Alexa Fluor 647-conjugated (ab150075, Abcam) secondary antibodies were used for labeling. The cells were imaged with a laser scanning confocal microscope. The average fluorescence intensity of 20 representative cells was obtained for quantitative analysis.

IHC staining was performed according to a previously described process [8 (link)]. Briefly, the mice were transcardially perfused with 0.9% saline, followed by 4% paraformaldehyde (PFA). The brains were removed and post-fixed in 4% PFA for 24 h at 4 °C and then dehydrated in 15% sucrose for 24 h, followed by 30% sucrose for 48 h. The brains were cut into coronal sections (25-μm thickness) with a rotary frozen slicer (CM1950, Leica, Germany) and stored in a cryoprotectant. The sections were washed three times with 0.3% Triton X-100 in phosphate-buffered saline (PBST). Each section was blocked with 5% goat serum in 0.3% PBST for 2 h at room temperature and then incubated overnight with primary antibodies at 4 °C. After being washed with 0.3% PBST, the sections were incubated with AlexaFlour® 488 secondary antibody or AlexaFlour® 594 secondary antibody for 2 h at room temperature. The primary antibodies used in this study were anti-beta amyloid (dilution: 1:400, CN:60342–1-Ig, Proteintech), anti-NeuN (dilution: 1:500, CN: ab104224, Abcam), anti-GFAP (dilution: 1:400, CN: ab7260, Abcam), anti-CD68 (dilution: 1:500, CN: ab237968, Abcam), and anti-Iba-1 (dilution: 1:1000, CN: ab178846, Abcam). Neuronal density was measured using ImageJ software and quantified as the ratio of the area occupied by neurons to the image area (1024 × 1024 pixels) at the same threshold.