2.7. Iterative Indirect Immunofluorescence Imaging

Iterative indirect immunofluorescence imaging (4i) is a multiplex imaging technique, recently developed by Gut et al. for in vitro samples [27]. We adapted this technique for multiplex staining of 2 µm FFPE sections, following the published protocol. In brief, FFPE tissue was processed for IF staining, as described above. HIAR was performed in Tris-EDTA buffer at pH 9.0 using a pressure cooker for 10 min. After HIAR, a staining chamber was attached to the slide. All following buffers were prepared and applied as published by Gut et al.: conventional blocking solution (1% BSA in PBS), 4i blocking solution (1% BSA and 150 mM maleimide in PBS), elution buffer (0.5M L-glycine, 3M urea, 3M guanidinium chloride and 70mM TCEP-HCl in ddH₂O, pH 2.5) and imaging buffer (700 mM N-acetyl-cysteine in in ddH₂O, pH 7.4). Sections were washed 6 times with ddH2O, and elution buffer was applied 3 times for 10 min. Elution buffer was removed, and 4i blocking solution was added for 1 h. Samples were washed 6 times with PBS, and primary antibodies in conventional blocking solution were incubated for 2 h. Samples were washed 6 times with PBS, and secondary antibodies and Hoechst 33342, diluted in conventional blocking solution, were incubated for 45 min. Thereafter, samples were washed 6 times with PBS and 1 time with ddH₂O. Imaging buffer was added, and defined regions of interest were imaged, as described in the next paragraph, under microscopy. Slides were stored in imaging buffer overnight at 4 °C. All other steps were performed at room temperature. These procedures were repeated for each antibody until required IF-plexity for the sample was reached. Image registration was performed using the Fiji ImageJ v1.52 descriptor-based registration plugin and Hoechst 33342 staining for registration. After registration, fluorescence intensities were manually adjusted for proper labeling of positive cells (excluding nonspecific background and auto-fluorescence signals) and QuPath image analysis software was used for cell segmentation and image analysis as described under histological analysis [28].