S1961

The primary antibodies used in this study include PD-L1 (22C3) (Dako, M3653, 1:50). The immunohistochemistry staining of PD-L1 (22C3) was performed on a Dako Autostainer Link 48 platform with the Dako K8002 detection kit, and the signals were amplified with a mouse linker (contained in the kit) and the signals enhanced with a DAB enhancer (Dako, S1961); the staining procedure was set similar to the FDA-approved PD-L1 (22C3) pharmDx staining procedure. All immunohistochemistry (IHC) sections were evaluated by two pathologists independently. PD-L1 ≥1% on tumor cells were defined as positive.

Formalin-fixed paraffin-embedded biopsy sections were deparaffinized and boiled in citrate buffer (S1699, Dako, Carpinteria CA). Endogenous peroxidase was quenched with 3% hydrogen peroxide, followed by incubation with anti-TSLP antibody (rat monoclonal GNE01.12F3.B5.4D11, Merck Research Labs, Palo Alto CA), and incubations with biotinylated secondary antibody (712-066-153, Jackson Immunoresearch, West Grove PA), ABC Elite (PK 7100, Vector, Burlingame CA), DAB chromogenic substrate (K3468, Dako) and enhancer (S1961, Dako). Sections were counterstained with Mayer’s Hematoxylin (Poly Scientific, Bay Shore NY), dehydrated, and cover-slipped.
TSLP content was evaluated by a pathologist (BJW) blind to molecular and clinical data on a scale of 0 (no TSLP present) to 3, based on intensity of the stain. Scoring was perfomed on 5 non-EoE, 5 inactive EoE, and 5 active EoE patient biopsies.