To begin, obtain previously prepared formula and fixed and paraffin embedded lung cancer tissue sample slide. Incubate it at 65 degrees Celsius for five minutes. Perform dehydration by immersing the slide in xylene, followed by ethanol solutions of decreasing concentrations.
Then immerse the slides in sterilized water three times for one minute each. For antigen retrieval, place the slide in a staining and repair box. Fill it with immunohistochemical antigen repair buffer working solution of pH six or nine.
Cover the box with a lid, and heat it in a microwave oven for eight minutes at 100%power. Then cool the slide to room temperature. Cover the tissue with a blocking solution, and then incubate in a humidified chamber at room temperature for 10 minutes.
After removing the blocking solution, cover the slide with the primary antibody working solution, and place it in a humidified chamber. Then incubate this chamber for one hour at 37 degrees Celsius. Next, wash the slide with wash buffer working solution for two minutes.
Add polymer horseradish peroxidase directly dropwise onto the slide, and incubate it for 15 minutes at room temperature. After washing the slide, add fluorophore working solution directly dropwise onto the slide, and incubate it for 10 minutes at room temperature. Once the slide is washed, give microwave treatment as demonstrated previously.
When the slide is cooled to room temperature, wash it three times with double distilled water for one minute each. Next, dip the slide in the wash buffer working solution for two minutes, and perform antibody incubation, horseradish peroxidase, and fluorophore addition as demonstrated. For cell nuclear staining, cover the slide with DAPI working solution and incubate it in a humidified chamber for five minutes at room temperature.
Then wash the slides with double distilled water three times, one minute each. Remove water droplets from the slide. Add 10 to 20 microliters of anti-fluorescence quencher to the slide, and seal it with a microscope cover glass.
