Multiplex Cyclic Fluorescent Immunohistochemistry

The challenge for immunohistochemistry is to choose the species of primary and secondary monoclonal antibodies to avoid cross reactivity and obtaining better specificity, and the reputability. Standardization is necessary to optimize the antibody dilution ratio. Meanwhile, another challenge is to align the localization of zygote cell and the subcellular structures.

The use of this protocol meet the technical evolution of the distribution of three lymphocytic subpopulations, such as CD three positive, CD three, and CD eight positive, and CD 20 positive. Based on in C cell counts and the protein expression in conserved tissues. The level of lymphocytic infiltration can be observed too.

In our protocol, the parameter antibodies and the fluorescence-labeled secondary antibodies derive from different species are diluted as a mixture, making us efficient. This mixture also ensures a specific match between antibody and antigen. We intend to focus on tumor microenvironment reprogramming to brain metastasis of lung cancer.

And the intercellular interaction between tumor-infiltrating lymphocytes on the tumor cells. To begin, obtain primary lung tumor or lung cancer, brain metastasis tissue. Embed the tumor tissue in paired paraffin blocks and using a microtome.

Cut it into four micrometer thick sections. Place the sections in water. Choose the best one using tweezers and adhere it onto the polylysine coated slide.

To enhance tissue adhesion, incubate the slide in an oven at 65 degrees Celsius for 30 minutes. For dehydration, immerse the slide in xylene, followed by ethanol solutions of decreasing concentrations. Then wash it with the ionized water for three minutes.

Next, submerge the slide in sodium citrate buffer solution. Subject the slide to high heat and pressure in a pressure cooker, and let it cool to room temperature in distilled water for three minutes. Finally, wash the slide three times with PBS for five minutes each.

To begin, obtain previously prepared slides containing cancer tissue sections. Prepare working mixture of primary antibodies. Add the antibody complex onto the slide section and incubate for one hour at room temperature.

Then wash the slide three times with 0.1%TWEEN PBS for five minutes. Prepare fluorescent secondary antibody mixture. Add it drop wise on the slide and incubate it at room temperature for one hour.

Then perform a second round of primary antibody incubation at room temperature for one hour. Wash the slide three times with 0.1%TWEEN PBS for five minutes each, and using a filter paper, remove excess water from the perimeter of the tissue. Similarly, perform a second round of secondary antibody incubation, and wash as demonstrated earlier.

Now, add potassium permanganate reagent to the tissue after one minute, rinse it with running water for five minutes. Perform dehydration with increasing concentrations of alcohol. Finally, add DAPI to completely cover the tissue section and place a cover slip for multi-spectral imaging.

Multiplex immunofluorescence imaging indicated the presence of CD three positive, CD eight positive, CD 20 positive cells in lung cancer brain metastases tissue.