Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry

Multiplex fluorescent immunohistochemistry identifies multiple cell types and their locations in intact formalin-fixed paraffin-embedded tissue, allowing not only for cell identification but also cell to cell spatial analysis. The main advantage of this manual technique is the use of multiple antibodies of the same species without cross reactivity. This technique allows for an in-depth view into the interactions of immune and cancer cells in the tumor microenvironment.

When trying this technique for the first time, allow for approximately three to four days of staining and keep track of each step completely during the process. A weaker method of the staining process and multiplex design is critical to decrease common and expected pitfalls. Begin by deparaffinizing and rehydrating the slides.

Lay them in the hybridization oven with tissue side up and bake them at 60 degrees Celsius for an hour. Take the slides out of the oven and allow them to cool for five to 10 minutes in a vertical slide rack. Then use a slide staining set to treat the slides three times with xylene and once with 100%ethanol, 95%ethanol, and 70%ethanol for 10 minutes each.

Wash the rack of slides with deionized water and then fix the slides by submerging them in neutral buffered formalin for 30 minutes. Wash the slides with water for two minutes and proceed with antigen retrieval. Place the rack of slides into a heat-resistant box filled with pH 6.0 or pH 9.0 antigen retrieval buffer.

Cover the box with plastic wrap and secure it with a rubber band. Place the box into an inverter-equipped microwave on the edge of the rotating plate and heat the slides for 45 seconds at 100%power followed by 15 minutes at 20%power. Let the slides cool for 15 to 20 minutes after microwaving.

Meanwhile, prepare working solutions of antibodies and fluorophores. Prepare the primary antibody diluent by dissolving 0.5 grams of bovine serum albumen granules in 50 milliliters of TBST or 1X PBS. Dilute each primary antibody working solution to a previously determined optimized concentration.

Dilute each fluorophore in the fluorophore diluent at a concentration of one to 100. On the last day of staining, prepare the DAPI working solution by adding three drops of DAPI to one milliliter of TBST. Once the slides are cooled, remove them from the microwave and take off the plastic wrap.

Wash them with deionized water for two minutes followed by a two-minute wash with TBST. After the wash, dry the slide around the tissue with a delicate task wipe and trace around the outside of the tissue with a hydrophobic barrier pen, taking care to not touch the tissue or let it dry out. Place each slide in the humidifier chamber and add approximately four drops of blocking solution to the tissue.

Then incubate the slides for 10 minutes at room temperature and proceed with primary antibody application. Remove the blocking solution from each slide by tapping the side of the slide on a stack of paper towels and use a delicate task wipe to remove the remaining solution. Place the slide back in the humidified chamber, add approximately 200 microliters of the working primary antibody, and incubate for an hour.

After the incubation, wash the slides three times by submerging them in TBST for two minutes per wash. Dab off the remaining TBST from each slide and apply approximately three to four drops of secondary antibody. Incubate the slides in the humidified chamber for 10 minutes.

Wash the slides again with TBST and apply approximately 100 microliters of the fluorophore working solution. Return the slides to the humidified chamber and incubate for 10 minutes. Repeat the TBST wash and then microwave the slides according to the manuscript directions to remove the antibodies.

An important thing to remember during this multiple-day staining process is stopping after the microwaving step and leaving the slides in antigen retrieval buffer overnight, ensuring tissue and stain integrity. Remove the last antibody application with the antigen retrieval solution and wash the slides with deionized water followed by TBST for two minutes per wash. Repeat the staining steps for the rest of the antibody flurophore pairs and then proceed with DAPI application.

Apply approximately 150 microliters of working DAPI solution to each slide and incubate them in the humidified chamber for 10 minutes. After incubation, wash the slides with TBST for approximately 30 seconds and mount the cover slips. Once the mounting media has dried, apply clear fingernail polish at the four corners of the cover slip to secure it.

To analyze the monoplex, image the slides on the microscope with a 250 millisecond exposure using DAPI to focus. Evaluate each monoplex slide by looking at the fluorescence intensity of the stained marker and compare this intensity with the background. Use the slide position in the final multiplex if the stained marker intensity is at least five times higher than the background.

When staining the multiplex, choose the appropriate order for each antibody and assign each fluorophore to an antibody. Proceed with staining the multiplex as previously described and prepare a blank slide that receives antibody diluent, fluorophore diluent, and TBST instead of primary antibody, fluorophore, or DAPI. When ready to image the multiplex on the microscope, set the exposure to 250 milliseconds for all channels and capture each image using DAPI to focus.

Then use the analysis software to evaluate each multiplex by fluorescent false color and by pathology view, which will confirm the specificity of each marker. To ensure success of the multiplex assay, a monoplex assay has to be performed to determine the order of each antibody. For example, when Foxp3 is in the third position of the array, specific and robust staining and colocalization with CD3 is demonstrated.

In contrast, when Foxp3 is at the first position, nonspecific staining of Foxp3 occurs. Grainy and effuse areas of red are present on most of the slide, and fluorescent intensity of these areas is low. Another important step in this assay is DEPI counterstaining, which is the basis for cell identification and further analysis.

A clear contrast can be seen between images with working and nonworking DAPI. For the multiplex assay, beautiful composite images don't always reflect an accurate stain. While CD3 is visualized and shows specific staining, the pathology brightfield view of CD163 proves that the antibody is nonspecific.

An optimal multiplex image demonstrates specific staining in a composite image, and the CD3 and CD163 specificity is confirmed with the pathology brightfield view. Following this procedure, self-phenotyping and spatial analysis calculations can be performed to further analyze cell to cell interactions. This technique has paved the way for researchers to explore spatial patterning of cells and intact tissue, furthering our understanding of the tumor immune microenvironment.