Pano multiplex ihc kit

PANO Multiplex IHC kit (Panovue) was used to examine specific cell markers including CD3 (Abcam, 16669), CD8 (ZSGB‐Bio, ZA0508), CD11c (CST, 45581), Clec9a (Abcam, ab223188), STING (CST, 13647). After the primary antibody was applied, the slides were incubated with secondary antibodies, followed by staining with fluorochrome (fluorochrome:tyramide signal amplification = 1:100). Next, the slides were subjected to microwave heat‐treated antigen retrieval in EDTA buffer (pH = 9.0) and then blocked with goat serum, followed by incubation with the next primary antibody. Nuclei were stained with DAPI after all of the antigens had been labeled. The stained slides were scanned using the Polaris Automatic Digital Slide Scanner (Akoya Biosciences, USA) which captured the fluorescence excitation spectrum at different wavelengths (480, 520, 620, 690, and 780 nm) within the proper exposure time. Multiple scans were combined to build a panoramic image. The images were analyzed and reconstructed images of sections by HALO image analysis software (lndica Labs, USA).

Tissue microarrays (TMAs) were constructed with FFPE blocks of archived tumor specimens of the recruited 98 ESCC patients. Two 1-mm cores from representative areas of each tumor sample were punched and arrayed onto a recipient paraffin block. Tissue sections (4 µm thick) obtained from TMA blocks were subjected to multiplex fluorescent immunohistochemistry (mfIHC) staining using the PANO Multiplex IHC kit(0004100100, Panovue, Beijing, China) to examine specific cell markers including CD11c (ab52632, Abcam, Cambridge, UK), CD45RO (55618, Cell Signaling, Danvers, MA, USA), CD68 (ZM0060, ZSGB-Bio), panCK (4545, Cell Signaling), and PD-L1 (13684, Cell Signaling) in panel A and CD4 (BX50023, BioLynx, Brockville, ON, Canada), CD8A (70306, Cell Signaling), CD56 (3576, Cell Signaling), FoxP3 (320202, BioLegend, San Diego, CA, USA), and granzyme B (ab4059, Abcam) in panel B. Different primary antibodies were sequentially applied, followed by horseradish peroxidase-conjugated secondary antibody incubation and Tyramide signal amplification (TSA). After each TSA operation, the slides were microwave heat-treated. Nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI, D9524, Sigma-Aldrich, St. Louis, MO, USA) after all the human antigens have been labeled (Pan et al., 2021 (link)).

The compositions of major immune cells in TLSs of ESCCs were examined on FFPE ESCC slides using mfIHC and the PANO Multiplex IHC kit (Panovue, BJ, China) with antibodies for CD20 (Abcam, Boston, MA), CD21 (Abcam), Ki67 (Abcam), CD4 (Abcam), CD8 (ZSGB‐BIO, Beijing, China), LAMP3 (Abcam) and 4ʹ‐6ʹ‐diamidino‐2‐phenylindole (DAPI) as described previously.⁷ The stained slides were scanned using a Vectra Polaris Automated Quantitative Pathology Imaging System and batch analysed with HALO imaging analysis software (Indica Labs, Corrales, NM). Each TLS was segmented into a B cell zone (CD20+ cell and CD21+ cell‐clustered region) and a T cell zone (CD4+ cell and CD8+ cell‐clustered region). Cells in TLSs were phenotyped as B cells (CD20+), FDCs (CD21+), proliferating cells (Ki67+), CD4+ T cells (CD4+), CD8+T cells (CD8+) and mature DCs (LAMP3+). The intensity for each marker was recorded. The intratumoural infiltration of CD8+ T cells, CD4+ Th cells, Treg cells, memory T cells, natural killer cells, DCs and macrophages were evaluated on ESCC tissue microarrays as previously described.⁷

Multiplex immunohistochemistry modified for adherent C-33A and HeLa cells was performed to identify the protein expression and localization using the PANO Multiplex IHC Kit (0001100100, Panovue, Beijing, China).

To explore the expression and distribution of SPP1 in PDAC and normal pancreatic tissues, formalin-fixed paraffin-embedded (FFPE) sections from cancerous and adjacent normal tissues were subjected to multiple IHCs using PANO Multiplex IHC kit (Panovue, Beijing, China, cat# 0079100100). Briefly, sections were deparaffinized in fresh xylene, rehydrated in a decreasing concentration of ethanol, then washed three times with PBS. The antigen retrieval was performed with the microwave heating method and cooled down for at least 10–15 min in an ice-water bath. Blocking was performed with a blocking solution (Panovue, China, cat# 0018001120) for 10 min at room temperature, followed by incubation with a primary antibody for 30 min, a secondary antibody for 10 min, and TSA Opal fluorophores for 10 min. Antigen retrieval, blocking, primary and antibody incubation, TSA Opal fluorophore staining were repeated for each marker. Finally, all sections were counterstained with DAPI (Sigma-Aldrich, USA, cat# D9542) for 5 min and mounted. Sections were scanned using the panoVIEW VS200 (Panovue, Beijing, China). Images captured and quantitative analysis were conducted using the HALO software. The following primary antibodies were used: SPP1 (Abcam, Cambridge, UK, cat# ab214050), CD68 (ZSGB-Bio, Beijing, China, cat# ZM0060), and PanCK (CST, Boston, MA, USA, cat# 4545).