Optimized Workflow for Iterative Bleaching Extends Multiplexity Imaging of Highly Autofluorescent Clinical Samples

[Narrator] Using spatial proteomics, named Nature Methods' 2024 Method of the Year, the precise locations of immune cells along with cell-cell interactions can be mapped within tissues. This reveals spatial patterns linked to clinical outcomes. Building complex antibody panels and imaging tissues with high endogenous fluorescence present significant time, resources, and expertise challenges for IBEX and other fluorescence microscopy techniques. Using IBEX, tumor-specific features were identified in high-risk follicular lymphoma patients, and with Human Cell Atlas colleagues. a comprehensive spatial map of the human thymus was created. Little is known about the immune cell composition, spatial interactions, and cytokine production differences across diverse mycobacterial pulmonary pathologies. This protocol bridges this gap. This protocol offers a low-cost, adaptable photoirradiation method to significantly reduce broad-spectrum tissue autofluorescence, especially in difficult FFPE samples, while preserving antibody signal for spatial analysis. To begin, immerse the slides with tissue in PBS after completing antigen retrieval. Remove one slide from the PBS and use a lint-free wipe to carefully dry off all excess buffer without touching the tissue. With a hydrophobic pen, draw a border around the tissue section on the slide. After repeating the process for the remaining slides, let the hydrophobic barriers dry for 10 minutes. Then, using a lint-free wipe, wick off the PBS from the tissue section without touching the tissue directly. Now add 200 microliters of blocking buffer on the slide and close the moisture chamber. Place the chamber in a non-heating scientific microwave with a temperature regulating mechanism and run the previously established program five cycles. During the blocking incubation, prepare the primary antibody-staining solution 1 with hooks and the blocking buffer containing Fc block. Combine the antibodies and gently mix the cocktail. When the microwave cycle completes, remove and open the slide chamber and wick off the blocking buffer from the slide without touching the tissue. Next, add 200 microliters of primary antibody-staining solution 1 to the slide. Return the chamber to the microwave and execute the primary antibody program again for approximately 30 minutes. Once the primary antibody labeling is complete, hold the slide vertically. To wash the slide, pipette 1,000 microliters of PBS onto the tissue, allowing it to run off. Wick off the excess PBS and fix the slide with 1% paraformaldehyde for 10 minutes at room temperature in the humidity chamber. After fixing, wash the slide thoroughly with 1,000 microliters of PBS three to five times and leave a layer of PBS on the tissue until the photoirradiation step. To prepare the photoirradiation box, gather a 150-watt LED lamp, a 40-watt RGBW flood LED lamp, a large plastic container with a capacity of 75.71 liters or more, fresh PBS, and a Petri dish. Now fill the Petri dish with 1x PBS to completely submerge the slide. Carry out the photoirradiation procedure in a cold room to minimize heat from the lamps. Then place the slide in the Petri dish, ensuring it is fully submerged in PBS. Next, place the 40-watt lamp directly above the Petri dish with the light source facing downward toward the slide and set the lamp to the red light mode. Finally, turn on both the 150-watt and 40-watt lamps and cover the entire setup with the plastic container lid. After two hours, switch the lamp to green light and incubate for 16 hours. The brightest fluorophores compatible with the dye inactivation protocol were identified and paired with lowly expressed markers to enhance signal detection. Autofluorescence was reduced significantly after photoirradiation, and at longer imaging wavelengths. Directly conjugated antibodies targeting highly expressed structural markers, alpha-smooth muscle actin and pan cytokeratin, replaced in the near infrared channel at 750 nanometers. Background signal was computationally subtracted using an unstained reference image and SimpleITK arithmetic, and true signals were enhanced through thresholding. Whole slide imaging of IBEX-stained sections revealed granulomas with necrotic cores and CD15-positive neutrophils. Mycobacterium tuberculosis or nontuberculous mycobacteria were detected near the granuloma necrotic core using anti-antigen 85B antibody. Granuloma associated lymphoid tissue contained CD20-positive B cells, CD4-positive T cells, a few CD8-positive T cells and CD45 labeling of all immune cells in the lung. IBEX imaging also enabled visualization of lung anatomical features, including bronchial epithelial cells, arterial smooth muscle, and CD68-positive macrophages.