Endogenous peroxidase

As described previously with modifications, paraffin sections of cerebral arteries were made. The sections were dewaxed with xylene and dehydrated through graded alcohol. Antigen retrieval was performed by pressure cooking in 0.01 M citrate buffer for 20 min. And then, the sections were incubated in endogenous peroxidase (DAKO) and protein block buffer to block endogenous peroxidase activity. After, the sections were incubated with primary antibodies at 4°C overnight. Next, the slides were incubated with a horseradish peroxidase-labeled streptavidin solution for 20 min at room temperature. Sections were finally stained with 3,3-diaminobenzidine tetrahydrochloride (DAB) and counterstained with hematoxylin. After dehydration and sealing, they were observed under the microscope. Semiquantitative analysis of tissue immunoreactivity was done.

The carotid was fixed in 4% paraformaldehyde overnight, and then processed, embedded in paraffin, and sectioned at 4 μm. The deparaffinized, rehydrated were microwaved in citrate buffer for antigen retrieval. Sections were incubated in endogenous peroxidase (DAKO) and protein block buffer, and then with primary antibodies indicated overnight at 4 °C. Slides were rinsed with washing buffer and incubated with labelled polymer-horseradish peroxidase-antimouse/antirabbit antibodies followed by DAB + chromogen detection (DAKO). After final washes, sections were counterstained with hematoxylin. All positive staining was confirmed by ensuring that no staining occurred under the same conditions with the use of non-immune rabbit or mouse control IgG.

Cell suspensions and tissue samples (spleen, liver, and tumor) were fixed in 5% formalin, embedded in paraffin (FFPE), and cut into 1.5 µm slices. Specimens were deparaffinized followed by rehydration in a descending ethanol gradient (100%, 80%, 70%). Antigen retrieval was done with Tris/EDTA buffer (pH 9) at 121 °C for 5 min using the decloaking chamber. The slides were cooled down for 10 min in a water bath. After blocking endogenous peroxidase (Dako, Hamburg, Germany), the slides were washed in TBS-T for 5 min and the primary antibody PD-L1 (clone E1L3N, Cell Signaling, Danvers, MA, USA) was applied in appropriate dilution (1:200) for 1 h. Afterwards, the sections were washed in TBS-T and the secondary antibody was applied for 30 min at room temperature. The sections were washed again and incubated for 10 min with DAB plus substrate-chromogen solution (Dako, Santa Clara, CA, USA). The specimens were rinsed with distilled water and counterstained with hematoxylin for 2 min. Then the sections were rinsed in tap water (5 min), distilled water (1 min) and, dehydrated in ascending ethanol gradient (70%, 80%, 100%). After 2 × 5 min cleaning steps the specimens were cover-slipped with xylene containing mounting medium.