Figure 3.

Probe validation on FFPE cells and tissue. PBMCs were either stimulated (PMA 500 ng/mL, ionomycin 1 µg/mL for 4 hr) or not stimulated. (A) ISH was performed on FFPE PBMC cell blocks with a ratio of 100:0, 50:50, 25:75, and 0:100 of stimulated to unstimulated PBMCs. Representative microphotographs: after stimulation, the TNF-α ISH signal was very strong and formed clusters with a cytoplasmic-like signal. Scale bar = 20 µm. Therefore, (B) ISH signal was quantified and expressed as the percentage of TNF-α⁺ and IFN-γ⁺ cells. The percentage of TNF-α⁺ and IFN-γ⁺ cells decreased with the decreased ratios of stimulated to unstimulated PBMCs. (C) Stimulated and unstimulated PBMCs were analyzed by flow cytometry for TNF-α and IFN-γ; stimulation leads to increased number of TNF-α⁺ and IFN-γ⁺ cells. (D) ISH assay validation on human FFPE tonsil tissue and chromogenic ISH for UBC, DapB, TNF-α, and IFN-γ were performed on 2-µm-thick human FFPE tonsil sections. UBC (positive control) has a high-abundance signal, whereas no signal for the bacterial gen DapB was observed in human FFPE tonsil tissue. Besides cells with low mRNA expression of TNF-α and IFN-γ, we also observed cells with high amount of mRNA signal (black box) in the tonsil. Scale bar = 50µm. Abbreviations: FFPE, formalin-fixed paraffin-embedded; IFN-γ, interferon-γ; ISH, in situ hybridization; PBMCs, peripheral blood mononuclear cells; PMA, phorbol 12-myristate 13-acetate; TNF-α, tumor necrosis factor-α.