Dako autostainer link 48 system

TMA slides were deparaffinized in xylene and rehydrated in graded ethanol series. Subsequently, a 3% H2O2 solution was used to quench endogenous peroxidase activity for 15 minutes. Heat-induced epitope retrieval was conducted using citrate acid buffer at pH 6.0 in a microwave oven for 10 minutes. To prevent nonspecific antibody binding, the slides were then incubated with 5% normal goat serum for 1 hour at room temperature. Next, a T-Cell Exhaustion Marker Antibody Panel (ab254018, Abcam) containing mouse monoclonal anti–PD-1 (1:200) and recombinant rabbit monoclonal anti–CTLA-4 (1:500), anti–TIM-3 (1:500), anti–LAG-3 (1:200), and anti-TIGIT (1:500) were applied to TMA sections at 4°C overnight. According to the manufacturer’s instructions, the sections were then stained with a Dako REAL EnVision Detection System (horseradish peroxidase; HRP, Rabbit/Mouse, Agilent) for 30 minutes at room temperature, followed by 3,3-diaminobenzidine coloration and hematoxylin counterstaining. Finally, the sections were dehydrated in a graded ethanol series and xylene. Dako PD‐L1 IHC 22C3 pharmDx assay (Agilent, Carpinteria, CA), as the first FDA‐approved PD‐L1 IHC companion diagnostic, was used for IHC staining of PD-L1 on a Dako Autostainer Link 48 system (Agilent, Carpinteria, CA) with an EnVision FLEX visualization system according to the manufacturer’s instructions.

Tissue sections were freshly cut to 4 μm sections, mounted on Fisherbrand
Superfrost Plus Microscope Slides (ThermoFisher), then dried at 60°C for 1 h.
IHC staining was carried out on a Dako Autostainer Link 48 system (Agilent
Technologies) using a Dako PD-L1 IHC 22C3 pharmDx kit (Agilent Technologies)
with an EnVision FLEX visualization system, and then counterstained with
hematoxylin according to the manufacturer’s instructions. The expression of
PD-L1 protein was quantitated using a combined positive score (CPS), which was
calculated as the number of PD-L1-stained cells (tumor cells, lymphocytes, and
macrophages) divided by the total number of viable tumor cells, multiplied by
100. A tumor specimen with a CPS ⩾1 was considered positive for PD-L1
expression.

Tissues were freshly cut into 4-μm sections, mounted on Fisherbrand Superfrost Plus Microscope Slides (Thermo Fisher Scientific), and then dried at 60°C for 1 hour. IHC staining was carried out on a Dako Autostainer Link 48 system (Agilent Technologies) using the Dako PD-L1 IHC 22C3 pharmDx Kit (Agilent Technologies) with the EnVision FLEX Visualization System and counterstained with hematoxylin according to the manufacturer's instructions. PD-L1 protein expression was determined using CPS, which was the number of PD-L1–stained cells (tumor cells, lymphocytes, and macrophages) divided by the total number of viable tumor cells and multiplied by 100. The specimen was considered to have PD-L1 expression if CPS ≥ 1.