Cy3 tsa

Paraffin-embedded kidney sections were used to assess the co-localization of zonula occludens-1 (ZO-1) and GSDMD-N. First, the sections were subjected to microwave-based antigen retrieval using ethylene diamine tetraacetic acid antigen retrieval solution (pH 8.0), followed by tyramide signal amplification (TSA). Briefly, the following steps were performed: 1) incubation of sections with anti-GSDMD-N (1:200; Abcam; EPR20829-408) at 4°C overnight, 2) incubation with horseradish peroxidase (HRP) (1:500; Servicebio)-conjugated secondary antibody for 50 min at room temperature, 3) reaction with CY3-TSA (Servicebio, China) for 10 min in the dark, 4) removal of nonspecific binding antibodies by microwave treatment, 5) incubation with anti-ZO-1 (1:200; Servicebio) at 4°C overnight, 6) incubation with HRP (1:500; Servicebio)-conjugated secondary antibody for 50 min in the dark, 7) reaction with fluorescein isothiocyanate-TSA (Servicebio; China) for 10 min in the dark, and 8) staining with diamidine phenyl indole solution for 10 min. The images were captured by fluorescence microscopy using excitation wavelengths of 330–380 nm (blue), 510–560 nm (red) and 515–555 nm (green). The proportion (%) of positively stained glomerular area across three fields of view was analyzed using IPP. The results were confirmed by a professional pathologist.

Paraffin-embedded kidney sections were used to assess colocalization of caspase-1 and GSDMD or NLRP3. First, sections were subjected to microwave-based antigen retrieval. Then, tyramide signal amplification (TSA) was performed as previously described (Faget and Hnasko 2015 (link)). Briefly, the following steps were performed: 1) incubating sections with anti-NLRP3 (1:1,000, Proteintech) or anti-GSDMD (1:500, Proteintech) at 4°C overnight, 2) incubating with horseradish peroxidase (HRP)-conjugated secondary antibody for 50 min at room temperature, 3) reacting with CY3-TSA (Servicebio, China) for 10 min in the dark, 4) removing nonspecific binding antibodies by microwave treatment, 5) incubating with anti-caspase-1 (1:50, Proteintech) at 4°C overnight, 6) incubating with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Servicebio, China) for 50 min in the dark, and 7) staining with DAPI solution for 10 min. Images were captured by fluorescence microscopy using excitation wavelengths of 510–560 nm (red) and 465–495 nm (green).

We obtained the tissue microarray from the Outdo Biotech company (HOrg-C110PT-01, Shanghai, China) and the ethics was approved. Briefly, the paraffin sections of pan-cancer samples were deparaffinized and then were blocked with 3% H2O2 and 2% BSA after antigen retrieval. Anti-PDIA5 antibody (Rabbit, 1:100, Proteintech, China), anti-CD68 antibody (Rabbit, 1:3000, Servicebio, China), and anti-CD163 antibody (Rabbit, 1:3000, Proteintech, China) were sequentially applied, followed by horseradish peroxidase-conjugated secondary antibody (GB23301 and GB23303; Servicebio, China) incubation and tyramide signal amplification (TSA) (CY5-TSA, CY3-TSA, and FITC-TSA; Servicebio, China) incubation. And then, 4’,6-Diamidino2-phenylindole dihydrochloride (DAPI) was applied for the nuclei staining. The stained slides were visualized for multispectral images under the Pannoramic Scanner (3D HISTECH, Hungary). DAPI glows blue by UV excitation wavelength 330-380nm and emission wavelength 420nm in the fluorescence spectra. CY5, FITC, and CY3 glow pink, green, and red. The excitation wavelength was 608-648nm, 465-495nm, and 510-560nm, respectively, with an emission wavelength of 672-712nm, 515-555nm, and 590nm.

The paraffin-embedded liver tissue sections were cut into 10 μm coronal sections and used for immunofluorescence staining. These prepared sections were blocked with mouse serum and incubated with ADRP/perilipin2 (15294-1-AP, Proteintech, Wuhan) overnight at 4 °C, followed by fluorescence conjugated secondary antibody (Servicebio, Wuhan, China). The sections then added CY3-TSA (Servicebio, Wuhan) and were incubated in dark for 10min, followed by antigen retrieval with EDTA antigen retrieval buffer (1 mM EDTA, pH 8.0). Subsequently, these sections were incubated with the second primary antibody LAMP1 (ab208943, abcam), and followed by a fluorescence conjugated secondary antibody from Servicebio. The immunofluorescence was captured by using a Nikon Eclipse C1 microscope.

Paraffin sections of glioma tissues collected from Xiangya Hospital, Central South University, were deparaffinized. After antigen retrieval, sections were blocked with 3% H₂O₂ and 2% BSA. Different primary antibodies, CD68 (Mouse, 1 : 3000, AiFang Biological, Changsha, China), CD163 (Rabbit, 1 : 3000, Proteintech, Wuhan, China), were sequentially applied, followed by horseradish peroxidase‐conjugated secondary antibody incubation (PV6001, PV6002, ZSGB‐BIO, Beijing, China) and tyramide signal amplification (TSA; Fitc‐TSA, CY3‐TSA and 647‐TSA [Servicebio, Wuhan, China]). After labeling with the human antigens, nuclei were stained with 4′,6‐diamidino2‐phenylindole dihydrochloride (DAPI), and an antifade mounting medium was applied. Stained slides were scanned using the Pannoramic Scanner (3D HISTECH, Budapest, Hungary) to obtain multispectral images. Regarding fluorescence spectra, DAPI glows blue at a UV excitation wavelength of 330–380 nm and emission wavelength of 420 nm, CD163 glows red at an excitation wavelength of 594 nm and emission wavelength of 615 nm, and CD68 glows pink at an excitation wavelength of 608–648 nm and emission wavelength of 672–712 nm. Multispectral images were analyzed, and positive cells were quantified at a single‐cell level by caseviewer (C.V 2.3, C.V 2.0) and pannoramic viewer (P.V 1.15.3) image analysis software.

We obtained the tissue microarray from the Outdo Biotech company (HOrg-C110PT-01, total number of cases: 69 cases, total points: 110 points, Shanghai, China). The tissue microarray was approved by the Ethics Committee. Each tumor/normal tissue has three to eight cores (diameter 1.5 mm). The primary Abs were VSIR (Rabbit, 1:200, Proteintech, China), CD68 (Rabbit, 1:3000, AiFang biological, China), CD163 (Rabbit, 1:3000, Proteintech, China), CD8 (Mouse, 1:3000, Proteintech, China). The secondary antibody was horseradish peroxidase-conjugated secondary antibody incubation (GB23301, GB23303, Servicebio, China), and the tyramide signal amplification was TSA (FITC-TSA, CY3-TSA, 594-TSA, and 647-TSA (Servicebio, China)). Multispectral images were analyzed, and positive cells were quantified at single-cell levels by Caseviewer (CV 2.3, CV 2.0) and Pannoramic viewer (PV 1.15.3) image analysis software. Negative control procedures included the omission of the primary antibody.