Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

The high volume of biological information obtaining from the multiplex barcoding image analysis allows scientists to understand the tumor microenvironment better, and the special distribution within tumor cells. The main advantage of this method is that it gives us much more detailed information from the same tissue sample with the possibility of doing deeper phenotyping of individual cells. This method enabled customizable panels to study tumor microenvironment to potentially discover predictive biomarker in cancer tissue to use as new target treatment.

To begin the oligonucleotide conjugated antibody staining, soak the cover slips in 0.1%Poly-L-Lysine solution for 24 hours, at room temperature to prepare them for tissue placement and adherence. After draining the Poly-L-Lysine solution, wash the cover slips with ultrapure water for 30 seconds. Post washing, remove the cover slips from the ultrapure water and place them on a lint-free towel to dry overnight.

Place 5-micron-thick sections of the tissue of interest on the center of a Poly-L-Lysine-charged cover slip, and allow them to dry overnight. Preheat a cover slip holder in an oven at 60 degrees Celsius, overnight. Place the cover slip containing tissue sections in the preheated holder.

After baking it at 60 degrees Celsius for 30 minutes, check to ensure the paraffin has melted away from the tissue. Quickly place the cover slip holder containing the sample sequentially, in a series of solutions. Finally, place the cover slip holder in DEPC-treated ultrapure water for two rounds of five minutes each.

Prepare a humidity chamber by placing a water-soaked paper towel at the bottom of an empty pipette tip box. Fill a pressure cooker with water, enough to cover half the height of a 50 milliliter beaker. Place 5 milliliters of methanol per sample in a beaker at 4 degrees Celsius.

Dilute the required volume of AR9 buffer to 1X with diethyl pyrocarbonate, or DEPC-treated ultrapure water. Next, fill a 50 milliliter glass beaker with approximately 40 milliliters of the diluted AR9 buffer. Submerge the sample in the cover slip holder in the beaker, and completely cover the beaker with aluminum foil.

Place the aluminum foil-covered beaker in the water-filled pressure cooker, and cook at about 15 PSI for 20 minutes. Then remove the beaker and carefully unwrap the aluminum foil. Allow the beaker to cool at room temperature for about 10 minutes.

Remove the cover slip holder from the 1X AR9 buffer. Incubate it twice in DEPC-treated ultrapure water for two minutes. Meanwhile, retrieve the hydration buffer, the antibody diluent, and blocking solutions N, G, J, and S from the multiplexed imaging staining kit.

Place the cover slip sample in 5 milliliters of the hydration buffer twice, for two minutes each. Then place the cover slip sample in 5 milliliters of the multiplexed imaging antibody diluent block and incubate for 20 to 30 minutes at room temperature. During the incubation, prepare the antibody cocktail.

After incubation, place the cover slip in the previously prepared humidity chamber. Pipette 190 microliters of the antibody cocktail onto the cover slip sample, and incubate at room temperature for three hours. Next, wash the sample twice, each with 5 milliliters of the multiplexed imaging antibody diluent block for two minutes.

To fix the bound antibodies to the tissue, incubate the cover slips for 10 minutes in 16%formaldehyde diluted to 1.6%with storage solution, and wash them three times with PBS. After incubating the cover slips for five minutes in methanol, pre-cool to 4 degrees Celsius. Following the wash, incubate the cover slips again for 20 minutes, in 5 milliliters of the fixative reagent diluted with PBS, and wash them three times with PBS before storing.

Prepare the reporter master mix following the composition mentioned in the text. Fill a well in a 96-well plate with 245 microliters of a solution containing the reporter master mix and the specific barcoded fluorophores for that experimental cycle. The figure shows a reporter plate configuration used here for a carcinoma panel.

To protect the barcoded fluorophores, adhere a foil plate cover over the 96-well reporter plate, and place the plate within the multiplexed imaging instrument. Calibrate the focus of the imaging using the DAPI channel by placing a sample cover slip on the microscope stage and manually pipetting 700 microliters of a 1:1500 titration of a nuclear stain solution onto the tissue. To prepare the multiplexed imaging instrument, dilute 10X multiplexed imaging buffer to 1X using DEPC-treated ultrapure water, and fill reagent bottles with appropriate solutions or solvents.

Set all the microscope parameters, and select the regions of interest on the sample cover slip to be imaged. Enter the experimental design into the instrument manager software. Click the Experiment button in the control software, and in the Experiment Setup and Management window, click the New Template button.

Type the project name into the space next to the Project button, and enter the total number of cycles. Click on the Channel Assignment button, type the information for each cycle into the columns, such as the correct cycle, well numbers, Z-stack places, marker name, class, and exposure time for each cycle. Then start the experiment by clicking Start Experiment, and save the experiment.

Click the QuPath icon on the computer, and open the software. Drag the qptiff file to the viewer window. Click the brightness and contrast button to open the Brightness Contrast window.

Check or uncheck the marker in the selected column to show or hide the marker signal. Click on the zoom to fit button to zoom in or out of the area of interest. When viewed using image analysis software, the composite images showed all the markers or selected markers as desired.

The signal intensity, dynamic range, and spatial distribution of all the markers were revealed. This technique allowed the analysis of all 26 markers at the subcellular level in a single tissue section. Following the multiplex image analysis process, by informatic approaches can process and analyze the high dimensional single cell data to infer biological and clinical insight.

Multiplexing barcoding image analysis is a powerful methodology that enables scientists to profiling tumor tissue and answer their research question according the markers in the panels.