Figure 1.

PANINI enables sensitive multiplexed, strand-specific nucleic acid and protein detection in archival tissues

(A) An overview of the experimental workflow and analytical framework for PANINI. In short, tissue autopsy sections (4 SIV-infected and 2 SIV-uninfected rhesus macaques for this study) are subject to PANINI, which couples nucleic acid amplification and peroxidase-catalyzed hapten deposition with antibody-based detection of both nucleic acid and protein targets. Multiplexed images were then acquired using multiplexed imaging platforms such as MIBI and CODEX and computationally analyzed for a high-resolution understanding of host-pathogen tissue interactions in situ.

(B) Representative IF and MIBI images of positive control 3D8 and negative control CEMs cell pellet sections stained in the same experiment. Nuclear stains, DAPI for IF and Histone H3 for MIBI, are in blue and cyan, respectively; vDNA is in white.

(C) Quantification of 3D8 cells from (B) that are positive for vDNA signals.

(D) Cross-platform compatibility of PANINI. Top Left: standard immunofluorescence on COVID-19 positive nasal tissues for the SARS-CoV-2 spike mRNA (SARS-CoV-2, white), cilia marker acetylated tubulin (ACTUB, magenta), and epithelia marker pan-cytokeratin (PanCK, green). Top Right: Vectra Polaris on reactive lymph nodes and cytokine transcripts (CCL2, magenta; IL8, yellow), the B cell marker (Pax5, blue), and the macrophage marker (CD68, green). Bottom: CODEX on COVID-19 positive (left) and negative (right) lung samples for the SARS-CoV-2 spike mRNA (SARS-CoV-2, white), neutrophil marker (CD15, red), macrophage marker (CD68, blue), type II pneumocytic marker (MUC1, magenta), and epithelia marker pan-cytokeratin (PanCK, yellow).

(E) Adjacent sections from an SIV-positive lymph node. Both slides underwent PANINI treatment. For the top section, a fast red chromogenic substrate was used for vRNA (red) with hematoxylin. The bottom section was stained with a MIBI-compatible antibody cocktail. Markers shown here delineate specific cell lineages: CD3 (T cells, yellow), CD20 (B cells, purple), CD11b (monocytes, blue), CD21 (B cells and FDCs, white), and SIV vRNA (green).

See also Figure S1.