G1401

The synthetic peptides were labeled with fluorescein isothiocyanate (FITC) by ChinaPeptides (QYAOBIO) Co., Ltd. (Shanghai, China). Intracellular localization of peptides was performed according to previous studies [47 (link),49 (link)]. As described in Section 3.8, E. coli was cultured to reach a viable bacteria count of approximately 10⁷ CFU/mL. The FITC-labeled peptides were dissolved in double-distilled water or dimethyl sulfoxide (final concentration 0.25%) at a final concentration of 250 μg/mL. Two peptides were mixed in equal proportions at equal concentrations to obtain a final concentration of 125 µg/mL. E. coli was treated with the FITC-labeled peptides and incubated at 60 r/min and 37 °C for 3 h. The bacteria were collected by centrifugation and washed twice with 0.1 M PBS (pH 7.4) and the supernatant was discarded. The resulting bacteria were fixed with 4% paraformaldehyde for 30 min and centrifuged at 2000× g for 3–5 min. Then, the supernatant was discarded and the resulting bacteria were mixed with 400 μL DAPI and 100 μL PBS, left for 10–15 min in the dark, washed twice with PBS and resuspended. One drop of the bacterial solution was added to a microscope slide. After the mounting medium (G1401, Servicebio, China) treatment, LSCM (Nikon Eclipse Ti, Tokyo, Japan) was used to analyze the localization of the peptides in E. coli cells.

NPC cells were cultured in a six-well plate with glass slides. The cells were fixed with 4% Paraformaldehyde Fix Solution for 1h after adherence. After washing with PBS, the non-specific binding sites were sealed with goat serum at room temperature for 30 min. After absorbing goat serum with filter paper, the cells were incubated with primary antibodies at 4 °C overnight, washed with PBS for 3 times at the next day, and incubated with the secondary antibody at room temperature in darkness for 1 h, washed with PBS, stained with DAPI (Solarbio, C0065) for 3 min, and again washed with PBS. The anti-fluorescence quenching sealed tablet was then applied (Servicebio, G1401). Imaging was performed with a confocal laser microscope (Leica, Germany).

The brain hemispheres of PAP mice were fixed in 4% paraformaldehyde for 2 days, dehydrated in graded ethanol, embedded in paraffin wax and cut into 5 µm sagittal sections. For fibrillar Aβ staining, brain sections of mice were incubated in thioflavin S (S19293, Yuanye, Shanghai, China) solution for 8 min at room temperature, then washed with ethanol and distilled water. The slides were mounted in a fluorescence mounting medium (G1401, Servicebio, Wuhan, China), dried in the dark for 2 h, and protected with coverslips. The size of Aβ plaque was determined by manually tracing its diameter. A total of six microscopic fields of each section were examined, including three fields in the cerebral cortex and three fields in the hippocampus.

On day 18 post-immunization, rat eyeballs were cut into 5 μm slices for immunofluorescence detection after embedding in paraffin. Briefly, the slides were dewaxed, antigenically repaired with ethylenediaminetetraacetic acid citrate (pH 6.0) antigen repair buffer (G1202, Servicebio, Wuhan, China), blocked with 10% goat serum for one hour, and incubated overnight in diluted primary antibody (anti-rabbit Iba-1, A19776, 1:300, ABclonal, Wuhan, China). Secondary antibodies (goat anti-rabbit Alexa Fluor 488, AS053, 1:300, ABclonal, Wuhan, China) were then incubated for 60 min at room temperature and protected from light, and slides were washed three times, followed by 4′,6-diamidino-2-phenylindole nuclear staining (G1012, Servicebio, Wuhan, China). Sections were washed again and sealed with anti-fade blocker (G1401, Servicebio, Wuhan, China) and analyzed using laser scanning confocal microscopy (Leica, Wetzlar, Germany). Cell count was conducted using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

After aforementioned deparaffinage, dehydration, and antigen recovery, mice brain slices were washed with PBS for 3 × 5 min and blocked in 5% donkey serum containing 0.5% Triton X-100 for 40 min. Next, slices were incubated with primary antibody (listed in Table 1) at 4°C for 24–48 h. After incubation with secondary antibody at 37°C for 1 h, slices were washed with PBS for 3 × 5 min and stained with DAPI reagent (G1012, Servicebio) for 10 min at room temperature. Slices were sealed with anti-fluorescence quencher (G1401, Servicebio) and imaged using a scanning microscope (SV120, OLYMPUS).

The Olfr2 expression was performed by immunofluorescence (IF) analysis. Ana-1 cells or BMDMs were cultured on 14 mm round coverslips (BS-14-RC, Biosharp) in the 24-well plate (703001, Nest) and treated as described above. Cells were fixed with 4% PFA for 10min at RT. After washed three times with cold PBS, samples were incubated with 5% BSA (G1208, Servicebio) for 10min at RT and maintained in Olfr2 antibody (1:500, Thermo Fischer) at 4°C overnight. Subsequently, cells were washed three times and stained with secondary antibody (1:200, GB25303, Servicebio) for 1h at RT. DAPI (G1012, Servicebio) was used to stain nuclei in the dark for 10min. After washed three times, coverslips were sealed with anti-fluorescence quenching reagent (G1401, Servicebio) on glass slides. Images were captured by Zeiss scanning microscope and analyzed via ImageJ software.

The distribution of miRNA was identified by FISH. Briefly, lung tissue was fixed by 4% paraformaldehyde and cut into 4 μm sections. Then, these sections were pre-hybridized in 200 μL of prehybridization solution for 1 h at 37°C and then hybridized with 250 μL of hybridization solution containing FAM probe (6 ng/μl) overnight at 37°C. Mouse anti-DIG-HRP (G1401, Servicebio) and CY3-TSA (G3016-3, Servicebio) were added to the slides and incubated in a dark condition for 5 min after washing off the hybridization. Cells were then stained with DAPI (1:800) and incubated for 8 min in the dark after washing with wash buffer at 42°C. Finally, different fields were observed under a fluorescence microscope (Olympus, Japan) with FAM at 488 nm. The sequences of the probes are as follows: miR-590-3p: 5′-ACTAGCTTATACATAAAATTA-3′; miR-146a-3p: 5′-CTGAAGAACTGAATTTCAGAGG-3′; miR-140-5p: 5′-CTGTTTGTGGCTGGGCAGACGA-3′; miR-Let-7e-3p: 5′-GGAAAGCTAGGAGGCCGTATAG-3′; miR-21-3p: 5′-CTACCATCGTGACATCTCCATG-3′.