Cd20 mouse anti human antibody

Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, cut into sections, and placed on adhesion microscope slides. Sections were subjected to immunohistochemical (IHC) staining according to standard procedures. We performed the IHC by using CD3 rabbit anti-human antibody (Biolynx, BX50022), CD20 mouse anti-human antibody (Dako, M0755), and CD56 mouse anti-human antibody (Cell Signaling Technology, 3576S). The above primary antibodies were incubated at 4°C overnight followed by using the BOND™ Polymer Refine Detection Kit (Leica, DS9800) according to the manufacturer’s instructions. Whole slide scanning was performed using panoramic MIDI under a ×20 objective lens. Tumor and stroma recognition was performed using the “tissue classification” module of HALO tissue analysis software (Indica Lab), based on the tumor morphology.

Multiplex immunofluorescence staining was performed using PANO 4-plex IHC kit (cat 10001100100, Panovue). We performed the fluorescent dyes by using the CD20 mouse anti-human antibody (Dako, M0755), CD86 rabbit anti-human antibody (CST, 91882S), and FCRL4 rabbit anti-human antibody (Abcam, ab239076). Different above primary antibodies were applied, followed by horseradish peroxidase-conjugated secondary antibody incubation and tyramide signal amplification. The slides were microwave heat-treated after each TSA operation. Nuclei were stained with DAPI (SIGMA-ALDRICH, D9542) after all the human antigens had been labelled. To obtain multispectral images, the stained slides were scanned using the Mantra System (PerkinElmer, Waltham, Massachusetts, USA), which captures the fluorescent spectra at 20-nm wavelength intervals from 420 to 720 nm with identical exposure time; the scans were combined to build a single stack image.