Multiplexed Immunofluorescence Imaging to Analyze Immunotherapy-Resistant T Cell Subpopulations

After thawing, use a paper towel to carefully dry the slides around the tissue sections, and encircle the tissues with a hydrophobic barrier pen. When the barrier has dried, fix the samples in 100% acetone for 5 minutes, followed by 2 minutes of air drying. Then, wash the slides in TBS for 10 minutes.

Pretreat the slides with three drops of 0.1% avidin for 10 minutes in the dark. Then, tap or flick the slides to cover the tissue with the avidin solution. Add three drops of 0.01% biotin to each sample, and tap or flick the biotin across the samples, followed by a TBS wash, as just demonstrated. Then, block any nonspecific binding with 100 microliters of 5% normal serum in TBS for 30 minutes at room temperature.

At the end of the incubation, tap the slides to remove the excess serum, and incubate the slides in 100 microliters of the primary antibody cocktail of interest for 1 hour in a humidified chamber. Wash the antibody-labeled slides in TBST for 5 minutes.

After drying, label the cells with 100 microliters of the secondary antibody cocktail of interest for 30 minutes in the humidified chamber, followed by a 10-minute wash in TBS. Incubate the dried secondary antibody labeled slides with 100 microliters of the tertiary antibody cocktail of interest for 30 minutes, followed by a final 10-minute TBS wash.

At the end of the wash, dry the slides, and mount the tissues with the DAPI-supplemented mounting medium. Then, cover each slide with a cover slip, and let the mounting medium set for at least 2 hours.

To scan the slides, open the microscope software, and load the image-scanning protocol. Load the first slide onto the microscope stage. Then, in the control bar, click Set Exposure, and adjust the exposure time for each filter until a sufficient but not saturating signal is obtained.

Click Start in the upper panel, and open the lab ID folder. Enter the ID of the first slide, and click Next.

To acquire the overview of the slide under bright field light at a 4x magnification, click Monochrome Imaging and Find Specimen. To select the area of tissue to be scanned, press the Control key, and use the cursor to select or deselect the regions of interest to be scanned at the 4x magnification.

To obtain a fluorescent red-blue-green image of each region, click Low-Power Imaging. For high-power field selection, press the Control key, and select at least five regions of interest that correspond to the areas of the tissue that will be scanned at the 20x magnification.

Then, to obtain a multispectral image acquisition of each field, click High-Power Imaging, and click Data Storage to store the images in the appropriate lab ID folder.