CODEX Multiplex Imaging for Bacterial Spatial Mapping in Breast Cancer Tumors

Tissues were fixed with 4% PFA for 24 h, dehydrated, paraffin-embedded, and sectioned into 5-μm slices on Poly-L-lysine slides (stored at 4°C after air-drying).The 18-plex antibody panel included CD3 (Akoya #4550119), CD8 (Akoya #4250012), PanCK (Akoya #4450020), and immune markers (full list in Table S1), conjugated to Alexa Fluor 647/750/Atto 550 and stored in Akoya Storage Buffer.Slides were baked at 55°C for 1 h, dewaxed with Histochoice (VWR #H103-4L), and antigen-retrieved using Tris-EDTA (pH 9, Solarbio #C1038) in a pressure cooker.

Antibody cocktails (1:200 dilution) were applied overnight at 4°C (first round) and 3 h at RT (second round), followed by fixation with 1.6% PFA and methanol.Slides were imaged on a PhenoCycler-Fusion (Akoya) with DAPI (1 ms), AF647 (150 ms), and AF750 (150 ms) channels. Bacterial localization was analyzed by U-Net model-based single-cell segmentation of DAPI-stained nuclei, followed by quantification of DARMR probe fluorescence (excitation/emission 488/660 nm) in QuPath with positive signals defined as mean intensity > 3× background.