Isolation of Cells with Morphological and Spatial Information from Oral Submucous Fibrosis Samples by Laser Capture Microdissection

Our main research interests are the mechanisms of malignant transformation and the development of predictive models of oral potentially malignant disorders, such as oral leukoplakia and oral submucous fibrosis. Genomic, transcriptomic, proteomic, and other multiomic sequencing analyses and deep learning artificial intelligence models are used to study the progression from oral potentially malignant disorders to oral cancer. Current sequencing and multiomics analyses are mostly based on frozen samples, but oral potentially malignant disorders are mostly formalin-fixed paraffin-embedded tissue, often by biopsy, and there is a need to develop experimental techniques based on paraffin samples.

Paraffin tissue samples from oral potentially malignant disorders and squamous cell carcinoma are available in large numbers and from a variety of sources, making them a very valuable resource for study. Our protocol can help fully use these resources to be understand the mechanisms of disease development and malignant transformation. To begin, using a microtome, cut the formalin-fixed paraffin-embedded oral submucous fibrosis tissues into continuous sections of three and 10 micrometers thickness, then, unfold the sections in water.

Place three micron sections on the adhesion microscope slide, or slide A.and 10 microns on PEN membrane slide, or slide B.Place both slides on a hot plate at 60 degrees Celsius. After two hours, place slides A and B in xylene for 10 minutes, then hydrate the slides in graded ethanol concentrations for one minute in each concentration. Next, place the slides in ultra-pure distilled water for 30 seconds.

Soak slides in Harris Hematoxylin dye solution for 90 seconds. Again, wash the slides thrice in ultra-pure distilled water for 30 seconds each. After that, submerge the slides in div-hematoxylin solution for two seconds.

Return slides into ultra-pure distilled water for 30 seconds, repeating three times. Next, immerse slides A and B in 1%re-blue solution for two seconds. After washing the slides with distilled water, place them in an eosin solution for two seconds.

Again, immerse slides in ultra-pure distilled water for 30 seconds before dehydrating and increasing ethanol concentrations. Then, immerse slide A in xylene for three minutes. Finally, apply a drop of the mounting medium to slide A and cover it with a glass cover.

To begin, turn on the laser microdissection system and software. Place slide A containing stained three micron sections of oral submucous fibrosis tissues on the slide stage. Observe the histological morphology to identify the area of interest for laser microdissection.

Remove slide A and place slide B with the tissue side facing down on the slide stage. Next, click the unload button to push out the collection device. Insert a PCR tube into the collection device and ensure the tube is fixed, then click the okay button.

Select the cap by clicking the corresponding red circle, marking the collector device to tube caps, and wait until the selected circle turns green, then click the draw button and draw plus cut button and use the mouse to sketch the area of interest. Click on start cut to capture the area of interest, which will be collected by the cap. Then, press the lower button to ensure the captured sample is collected.

Click the specimen button to capture the next sample. Finally, click unload to unload the PCR tube with the captured sample. In this study, using laser microdissection of oral submucous fibrosis, tissues of epithelium and stroma with different patterns are captured.

Copy number alternations were present in epithelial samples, but not in stroma samples. The copy number alternations pattern in the dysplastic epithelium was observed in chromosomes three and eight, while copy number alternations were detected at a lower frequency in chromosome eight in the atrophic epithelium.