Cd45ro

The PANO Multiplex IHC kit (Panovue, Beijing, China) was used to examine specific markers of immune cells, including CD11c (Abcam), CD45RO (Cell Signaling), CD68 (ZSGB-Bio), panCK (Cell Signaling), and PD-L1 (Cell Signaling) in panel A, and CD4 (biolynx), CD8A (Cell Signaling), CD56 (Cell Signaling), FoxP3 (Biolegend), and granzyme B (Abcam) in panel B in two 4-µm sections from tissue microarrays blocks. Primary antibodies were applied, followed by horseradish peroxidase conjugated secondary antibody incubation. Then the slides were microwave heat-treated after tyramide signal amplification operation. Lastly, DAPI (Sigma-Aldrich) were subjected to stain the nuclei after all antigens had been labeled.
A Vectra Multispectral Imaging System (PerkinElmer) was used for getting images. One image per core was captured at 200x magnification and each multispectral image cube was performed by combining images obtained every 10 nm of emission light spectrum across the range of emission filter cube. Five filter cubes, including DAPI (440-680 nm), FITC (520-680 nm), CY3 (570-690 nm), CY5 (670-720 nm), and Texas Red (580-700 nm), were used for each image capturing.

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)).