Goat anti rabbit igg h l horseradish peroxidase hrp

HCC specimens and adjacent tissue specimens were cut into 4-µm paraffin-embedded sections, mounted on slides, deparaffinized with xylene and rehydrated with graded ethanol, and blocked with 3% hydrogen peroxide. Heat-induced antigen retrieval was performed using a steamer at 95°C. The primary antibody Rabbit Anti-YAP1 antibody (Abcam, MA, USA) was diluted at a ratio of 1:50. Incubation was performed at 4°C overnight, followed by incubation with a secondary antibody Goat Anti-Rabbit IgG H&L (horseradish peroxidase [HRP]) (Abcam) in 1:50,000 dilution at 37°C for 60 min. Diaminobenzidine was subsequently applied for 10 min. Finally, the sections were counterstained with hematoxylin, dehydrated, covered, and visualized. According to previous studies,12 (link),13 (link) the expression of YAP1 was determined by the density of positive cells and the intensity of staining. The density was scored based on the proportion of positive cells (0: none; 1: ≤10%; 2: 11–25%; 3: 26–50%; 4: >50%), and the intensity was scored based on the average staining intensity of positive cells (0: none; 1: weak; 2: intermediate; 3: strong). The final score was the product of the density score and intensity score, ranging 0–12. Patients were classified into two groups: YAP1 low expression (score: 0–3) and YAP1 high expression (score: 4–12).

Fluorophore-coupled mouse monoclonal antibodies (mAbs) against human CD8α eFluor 450, CD25-PerCP-efluor710/PerCP-Cy5.5, CD134 (OX-40)-APC, CD279 (PD-1)-PE-Cyanine7 and CD137 (4-1BB)-FITC were purchased from eBioscience (San Diego, CA); anti-CD3 (OKT3), anti-CD28 (clone: 15E8), anti-CD4-FITC, and anti-CD197 (CCR7)-FITC were from Miltenyi Biotec (Bergisch Gladbach, Germany); anti-CD3-APC (clone HIT3a) was from SIGMA-ALDRICH (St. Louis, MO); anti-T-bet-Alexa Fluor 647 (Clone: 4B10) and anti-CD45RA-APC were from BioLegend (San Diego, CA); anti-human IL-18/IL-1F4-Alexa Fluor 647 and recombinant human IL-18/IL-1F4 were from R&D Systems (Minneapolis, MN); rabbit anti-human IL-18, goat anti-rabbit IgG H&L-horseradish peroxidase (HRP), goat anti-rabbit IgG H&L-Alexa Fluor 488 and rabbit IgG monoclonal Isotype Control were from Abcam (Cambridge, UK); goat anti-rabbit IgG (H&L) highly cross-adsorbed-Alexa Fluor 488 was from Invitrogen (Waltham, Massachusetts, USA).