The primary antibodies used for immunohistochemistry (IHC) and multiplex immunofluorescence staining are presented in the online supplemental materials and methods . IHC staining of the cancer lesions and TMA slides was performed as described previously.31 (link) Multiplex immunofluorescence was performed using a PANO 7-plex IHC kit (Panovue, Beijing, China), according to manufacturer’s instructions. Different primary antibodies were sequentially applied, followed by horseradish peroxidase-conjugated secondary antibody incubation and tyramide signal amplification (TSA) using a TSA Fluorescence kit (Panovue, Beijing, China). After labeling with the human antigens, nuclei were stained with 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI). Stained slides were scanned using the PanoVIEW vs200 slide scanner (Panovue, Beijing, China), equipped with an Olympus 20×lens, to obtain multispectral images. Fluorescence spectra were captured at 20 nm wavelength intervals from 420 to 720 nm, with identical exposure time. Multispectral images were analyzed, and positive cells were quantified at a single-cell level by QuPath v0.2.0 image analysis software (Queen’s University Belfast, Northern Ireland, UK) on a PanoATLAS workstation.
Tsa fluorescence kit
Manufactured by Panovue
Sourced in China
The TSA Fluorescence kit is a laboratory equipment product used for fluorescence-based detection and analysis. It provides the core functionality of fluorescence-based assays, without interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
2 protocols using tsa fluorescence kit
1
Multiplex Immunofluorescence for Cancer Profiling
2
Multiplex Immunofluorescence for T Follicular Helper Cells and Tumor Cells
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Multiplex immunofluorescence (IF) staining for markers of T follicular helper cells (CD4 and BCL6) and tumour cells (Pan‐keratin) in tumour tissue samples was performed as described previously.70 The staining was obtained using PANO 7‐plex IHC kit (0004100100, Panovue, Beijing, China) and tyramide signal amplification (TSA) fluorescence kit (10021001050, Panovue, Beijing, China). The following primary antibodies were sequentially applied in accordance with the manufacturers’ recommendations: CD4 (ZM‐0418, Beijing Zhongshan Golden Bridge Biotechnology, Beijing, China), BCL6 (89369, Cell Signaling Technology, Massachusetts, USA) and Pan‐keratin (4545S, Cell Signaling Technology, Massachusetts, USA), followed by polymer horseradish peroxidase‐conjugated secondary antibody (10013001050, Panovue, Beijing, China) incubation. The nuclei staining was incubated by 4′‐6′ diamidino‐2‐phenylindole (DAPI, D9542, Sigma Aldrich).
The stained slides were scanned by Mantra System (PerkinElmer, Waltham, Massachusetts, USA). And the quantification of positively stained cells was performed using inform image analysis software (Version 2.4, PerkinElmer, Waltham, Massachusetts, USA).
The stained slides were scanned by Mantra System (PerkinElmer, Waltham, Massachusetts, USA). And the quantification of positively stained cells was performed using inform image analysis software (Version 2.4, PerkinElmer, Waltham, Massachusetts, USA).
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