Iterative Indirect Immunofluorescence Imaging (4i) on Adherent Cells and Tissue Sections

Abstract

Highly multiplexed protein measurements from multiple spatial scales using fluorescence microscopy recently emerged as a powerful way to investigate tumor microenvironments in biomedicine and the multivariate nature of complex systems' interactions. A range of methods for this exist, which either rely on directly labeling the primary antibody with oligonucleotides/rare metals or employing methods to remove fluorescence for cyclic acquisition. Here, we describe a protocol that uses off-the-shelf primary and secondary antibodies without further need for modification and only commonly available chemical reagents. The method harnesses the observation that antibodies can crosslink to bound epitopes during light exposure, thus preventing elution. By utilizing a simple oxygen radical scavenging buffer during imaging and by blocking free sulfhydryl groups before antibody incubation, the presented method can employ comparably mild conditions to remove bound antibodies from epitopes, which preserves sample integrity. Thus, with the stated minor modifications, it allows for a standard immunofluorescence imaging protocol in cyclic fashion, currently permitting staining of up to ~80 unique epitopes.

Keywords: Antibody; FFPE sections; Fluorescence microscopy; High-throughput imaging; Histology; Multiplexed imaging; Tissue imaging.

Figure 1.. Flowchart depicting the standard workflow of 4i

Figure 2.. Multiplexed images obtained by performing 4i.

184A1 cells were deprived of serum and growth factor for 16 h, and then stimulated with 100 ng/mL EGF for 5 min before fixation. Then, 4i was performed as described for the tissue culture cells. Representative composites of a 30-plex experiment are displayed with the stained epitopes indicated. The same field of view is shown in all composites. Scale bars: 20 μm.

Figure 3.. 4i is reproducible across acquisition cycles.

The top panel shows representative images of repeated staining against phosphorylated ERK in different cycles (01, 07, and 13) in 184A1 cells exposed to 100 ng/mL of EGF. The bottom panel shows the bivariate plot of quantified pERK intensities for each combination of indicated 4i cycles. Scale bars: 20 μm.

Figure 4.. Standard workflow of an antibody panel design experiment

Figure 5.. Images obtained during an antibody panel design experiment.

Lamin B is an example of an antibody that needs to be prioritized, due to altered signal in later cycles. The top panel depicts staining at the respective cycles with mock cycles (repeated elution but no antibody incubations) with both primary and secondary antibodies. Bottom panel depicts the residual signal after elution and staining with only secondary antibody.