Two mIF panels were developed to characterize the locally advanced NSCLC immune microenvironment. CD4 panel including Pan-CK, CD4, FoxP3, CD8, CD31, α-SMA, HIF-1α and DAPI. CD8 panel including Pan-CK, CD8, CD103, PD-1, TIM-3 and DAPI.
The procedure for mIF has been previously described (21 (link), 30 (link)–33 (link)). In summary, 3 μm serial sections obtained from TMA blocks were deparaffinized and rehydrated using a decreasing ethanol series. Antigen dretrieval was performed by using microwaves. A blocking buffer was then used to initiate protein stabilization and background reduction for 30-60 minutes at room temperature. Primary antibodies were applied and washed in 1×Tris-buffered saline containing 0.5% Tween 20. Isotype-specific HRP-conjugated antibodies and an Opal fluorophore (Akoya Biosciences). Finally, the nuclei were stained with DAPI (Akoya Biosciences) for 10 min. Antibodies and fluorophores used in the mIF procedures were detailed inSupplementary Table 1
The procedure for mIF has been previously described (21 (link), 30 (link)–33 (link)). In summary, 3 μm serial sections obtained from TMA blocks were deparaffinized and rehydrated using a decreasing ethanol series. Antigen dretrieval was performed by using microwaves. A blocking buffer was then used to initiate protein stabilization and background reduction for 30-60 minutes at room temperature. Primary antibodies were applied and washed in 1×Tris-buffered saline containing 0.5% Tween 20. Isotype-specific HRP-conjugated antibodies and an Opal fluorophore (Akoya Biosciences). Finally, the nuclei were stained with DAPI (Akoya Biosciences) for 10 min. Antibodies and fluorophores used in the mIF procedures were detailed in
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