Gb22303

A batch of three domes (50 μl) of cultured organoids was dissolved by manual disruption to initiate immunostaining.⁴⁴ After centrifugation (100 × g, 4°C, 5 min), sedimentary organoids were fixed in 4% paraformaldehyde solution in PBS for 30–60 min. To block and permeabilize organoids, 1 ml Triton X‐100 and 2 g BSA were added to 1 L PBS to prepare organoid wash buffer (OWB). The fixed organoids were permeabilized using OWB for 20 min. Primary antibodies (anti‐Ki‐67, 11‐5698‐82; anti‐E‐cadherin, 53‐3249‐82; Thermo Fisher; anti‐CK‐20, ab109111; anti‐MUC2, ab272692; anti‐lysozyme, ab108508; anti‐CHGA, ab254322; Abcam, Cambridge, UK) were incubated in OWB (1:100 dilution) at 4°C overnight in rotation (60 rpm).⁴⁵ After extensive washing, second antibodies (GB22301, GB22303; Servicebio) were incubated in OWB (1:200 dilution) at 4°C overnight on rotation (60 rpm). Nuclei (KGA215; KeyGEN BioTECH) and live/dead (KGAF001; KeyGEN BioTECH) staining was performed. Prepared organoids were transported into a glass bottom dish and observed using confocal microscopy (LSM900; Zeiss, Jena, Germany).

The rabbit carotid aneurysm tissue, prepared as described earlier, were de-paraffinized, rehydrated and subjected to antigen retrieval. The sections were then incubated with rabbit anti-MPO (1:100, Bioss antibody, bs-4943R) overnight at 4 °C, FITC-labeled goat anti-rabbit secondary antibody (1:100, Servicebio, GB22303)was added and incubated at room temperature for 30 min. DAPI (ZSGB-BIO, ZLI-9557) was added at room temperature for 10 min to delineate nucleus. The sections were imaged using a microscopic camera system (3DHISTECH, CaseViewer 2.4).

The tumor samples were deparaffinized by two changes of xylene (15 min each), followed by dehydration in a 100–75% ethanol series (5 min each) and a distilled water wash. The tumor tissue sections were subsequently immersed in EDTA antigen repair buffer (pH 8.0), and the antigen was recovered by microwaving (10 min). We then added 3% BSA to block nonspecific binding sites for 30 min. The slides were subsequently incubated with the primary antibody rabbit anti-mouse CD4 (1:200, #GB13064-2, Servicebio) or the primary antibody rabbit anti-mouse CD8 (1:200, #GB11068, Servicebio) overnight at 4 °C. The tissue sections were then rinsed in PBS (pH 7.4) and incubated with FITC-conjugated goat anti-rabbit IgG (H + L) (1:100, #GB22303, Servicebio) at room temperature for 50 min in the dark. The sections were incubated in DAPI solution for 10 min at room temperature, and immunofluorescent images were taken using a Nikon Eclipse C1 microscope (Japan) and using Image J to evaluate the positive rates of CD4 and CD8 proteins.

In brief, after formalin fixing, cells were permeabilized with 0.1% Triton X-100 in TBS for 5 minutes, followed by a 30-minute block with goat serum. Next, cells were subjected to incubation with the AAK1 antibody (1:50, PA5408, Abmart) for 1h and hatched with FITC-conjugated goat anti-rabbit IgG (1:100, GB22303, servicebio) at room temperature for 2 hours at 37°C. Subsequently, the nucleus was marked with DAPI (BL105A, Biosharp). Finally, the cells were captured using a fluorescence microscope (Olympus, Tokyo, Japan).

After dewaxing, the paraffin sections were subjected to antigen retrieval with citrate buffer (pH = 6). Then, sections were rinsed with phosphate buffer saline (PBS, Beyotime, Shanghai, China) thrice and blocked with goat serum (SP9002, ZSGB-BIO, Beijing, China) for 20 min at the room temperature. Next, sections were incubated with CD68, Iba-1, C3, and the GFAP antibody overnight at 4°C. Subsequently, sections were washed with PBS three times and incubated with FITC conjugated Goat anti-rabbit IgG (H + L) (1:100, GB22303, Servicebio, Wuhan, China) or Cy3-conjugated Goat anti-rabbit IgG (H + L) (1:100, GB21303, Servicebio) at 37 °C for 30 min. The nuclei were stained with DAPI (ZLI-9557, ZSGB-BIO) for 10 min at room temperature. Images were captured with a confocal microscope (LSM700; Zeiss, Oberkochen, Germany).

Fixed brain tissue was paraffin-embedded and sectioned, and then dehydrated and washed. The section was blocked with BSA for 30 min, incubated with primary antibody in PBST overnight at 4 °C. The section was then washed 3 × 5 min in PBST, and incubated with secondary antibody in PBST for 1 h at room temperature. Then, the section was incubated sequentially with DAPI staining solution and autofluorescence quencher. Finally, after three washes with PBS, the section was mounted with anti-fluorescence quenching mounting medium, and the mounted section was observed and imaged under CLSM. Primary antibody against tyrosine hydroxylase (Servicebio, validated by manufacturer, cat# GB11181, 1:1000 dilution) and secondary antibody goat anti-rabbit labeled with FITC (Servicebio, validated by manufacturer, cat# GB22303, 1:100 dilution) were used.