Opal polaris 7 color ihc detection kit

Tissue sections of the HCC patients were stained against Axl (1:50; R&D; #AF154), CD8 (1:200; Abcam; #ab4055), Granzyme B (1:100; Abcam; #ab4059), CD31 (1:100; Abcam; #ab28364), α-smooth muscle actin (1:100; Dako; #M0851), CD45 (1:100; BioLegend, San Diego, CA, USA; #B368502) and DAPI using the Opal Polaris 7 Color IHC Detection Kit (Akoya Biosciences; Marlborough, MA, USA). The stained sections were scanned using the Vectra Polaris^TM automated imaging system (Perkin Elmer; Hopkinton, MA; USA) and analyzed using HALO AI software version 3.6 (Indica labs; Albuquerque, NM; USA). The cells were classified according to their expression of DAPI and the respective marker. The relative cell count was obtained by normalizing the number of cells positive for both DAPI and the marker to the total number of cells positive for DAPI.

Formalin‐fixed and paraffin‐embedded normal colonic and polyp tissues from paediatric patients sectioned to 4 µm were used for histological evaluation. Haematoxylin and eosin staining was performed on each polyp sample. For IF staining, tissue slides were deparaffinized, rehydrated and processed to antigen retrieval. Primary antibodies for anti‐EpCAM (1:100, Invitrogen, Life Technologies, #14‐9326‐82), anti‐TGFBI (1:150, Invitrogen, #MA5‐32736) and anti‐LAMC2 (1:750, Invitrogen, #PA5‐109901) were used. The slides were then incubated with secondary antibodies for 10 min at room temperature and counterstained for nuclei with DAPI (2 µg/mL, Invitrogen, #D1306) for 15 min. The images were scanned with a laser scanning confocal microscope (Carl Zeiss Meditec AG). For mIHC analysis, primary antibodies against CD3 (1:5, MXB Biotechnologies, #MAB‐0740), CD19 (1:2000, Cell Signaling Technology, #90176), EpCAM (1:2000, Abcam, #ab223582), PECAM1 (1:5, MXB Biotechnologies, #MAB‐0720), CD11C (1:2000, Cell Signaling Technology, #45581) and α‐SMA (1:2000, Boster Biological Technology, #BM0002) were used. Multiplex IF staining was performed using the Opal Polaris 7 Color IHC Detection Kit (Akoya Bioscience, #NEL871001KT), and multispectral images were scanned with PhenoImager HT (Akoya Bioscience).

Letrozole (T1590) was purchased from TargetMol (Bellingham, WA, USA). CYP19A1 siRNA (sc-41498) and CYP19A1 antibody (sc-374176) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PD-L1 (13684) antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). PE-conjugated anti-CD3 antibody (555340), APC-conjugated anti-CD8 antibody (566852), PE-Cy7-conjugated anti-IFN-γ antibody (557643) were purchased from BD Biosciences (San Jose, CA, USA). FITC-conjugated anti-CD107a antibody (328606) and Zombie NIR (423105) were purchased from BioLegend (San Diego, CA, USA). Opal Polaris 7 Color IHC Detection Kit (NEL861001KT) was purchased from Akoya Bioscience (Menlo Park, CA, USA). Chitosan (CS, deacetylation 98%, Mw = 50 KDa) and sodium tripolyphosphate (TPP) were purchased from Sigma Chemical Co (St. Louis, MO, USA). Hyaluronic acid (HA) was purchased from Meryer Chemical Technology Co Ltd (Shanghai, China).