Image Acquisition After Multiplex Immuno-Staining for Distinguishing uNK Cell Subtypes in Endometrial Tissues

To begin, stain the mounted endometrial tissue sections with the desired antibodies. Turn on the imager and select appropriate fluorophores based on TSA dyes. Manually focus on an area of the slide that shows a fair exposure of each marker and use the auto-exposure feature to set the exposure time for that fluorophore.

To ensure unbiased sampling, randomly select the initial area for image acquisition with a focus on the luminal epithelial border. After capturing the initial field, move one field left or right to select subsequent areas, skipping one field between each image captured. Maintain the luminal epithelial border within the frame to capture at least five different areas to include around 10, 000 stromal cells.

TSA monoplex and multiplex staining revealed clear colocalization of NK cell markers, allowing the identification of distinct NK cell populations. The multiplex strategy used fluorescent dyes with distant wavelengths to avoid spectral overlap and ensure clear background, confirming signal specificity.