Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue

Our research focused on optimizing the mIHC protocol on paraffin section obtained from clinic sources. We aimed to effectively address autofluorescence and autofluorescence channel crosstalk issues in lung tissue. The current experimental challenges we are facing involve addressing the issues of ultrafluorescence and the channel crosstalk in lung tissue, thereby achieving successful what color fluorescence localization they need for labeled target cells and proteins.

Our highly efficient multiple antigen insight leveling method, which requires less than three hours per antibody, seamlessly integrates a multi-spectro emergent system and a specialized pathology software. This optimized mIHC protocol is easily adaptable for use in other research laboratories. 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 horse radish 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, horse radish 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. To begin, obtain previously prepared stained lung cancer tissue sample slides.

For automatic scanning of the stained slides, manually set the exposure time and scanning program for each fluorescence channel. Drag the multicolor fluorescence image file obtained from the original scan into the software and set the image type to Fluorescence. Click the Brightness contrast button and check the channels on the panel.

Click outside and then on the channel to select the fluorescence channel of interest. Drag the minimum display and maximum display to adjust the brightness and contrast of each channel. Once the view to be saved is determined, click on Show Slide Overview and Show Cursor Location to remove these two contents and keep the scale bar.

Click File followed by Export Snapshot and Current viewer content to export a PNG file. The non-small cell lung squamous carcinoma tissues showed that macrophages and CD8-positive T cells significantly infiltrated around and within carcinoma foci. HMGCS1 protein exhibited widespread expression in squamous carcinoma cell plasma.