This article describes a staining technique for examining cancer cell invasion through elastic fibers in tissue sections. The method involves a series of chemical treatments to visualize elastic fibers and differentiate between various tissue types under a microscope.
Elastic fiber staining helps examine cancer cell invasion beyond the peritoneal elastic lamina or the sub-mesothelial elastic tissue.
To begin, add potassium permanganate solution to a processed tissue section, followed by immersion in oxalic acid. The strong oxidizing property of the potassium permanganate-oxalic acid combination bleaches the natural pigment from the tissue section to prevent interference with the staining process by obscuring elastic fiber morphology.
Rinse the slide with water and ethanol to remove any residual bleaching agent. Next, incubate the specimen in an elastin stain that comprises an iron-hematoxylin complex. Elastin, the main elastic fiber component, has a strong affinity for iron-hematoxylin complexes.
Wash the slide with ethanol and water to remove any residual stain. Finally, treat the specimen with a counterstain containing picric acid and Ponceau S. The smaller picric acid molecules penetrate all tissue types but are firmly retained by the close textured muscle and cancer cells. In contrast, the larger Ponceau S molecules displace picric acid in the porous-textured collagen, causing collagen to be stained differently from the muscle and cancer cells.
When observed under a microscope, elastic fibers appear as blue-black filaments. Collagen stains red, while muscle and cancer cells appear yellow. Yellow cancer cells penetrating the blue-black elastic lamina confirm metastatic invasion.
First, use tissue paper to wipe off any excess solution on the slide or around the tissue. Place the slides in conditions that match room temperature and humidity. Add a few drops of potassium permanganate to each slide to oxidize them, making sure that the volume added is enough to cover the tissue section on each slide. After 5 minutes, rinse the oxidized slides with distilled water in Coplin jars, repeating the rinse two to three times, and using a new jar of water for each rinse. The tissue should have an observable brown color.
Now, add a few drops of oxalic acid to each slide to bleach them, making sure that the volume added is enough to cover the tissue section on each slide. After 5 minutes, rinse the bleach slides with distilled water, repeating the rinse two to three times, and using a new jar of water for each rinse. Wash the slides briefly in 95% alcohol.
Immerse the washed slides in elastin solution for 8 to 24 hours. Then, immerse the slides directly in 95% ethanol for 1 to 2 minutes to differentiate each tissue section well. Rinse the slides completely with distilled water.
Check microscopically for the blue-black elastic fibers staining and the gray background. It's better to slightly under differentiate the tissue since the subsequent Van Gieson's counterstain can also somewhat extract the elastic stain.
Counterstain the slides in a Van Gieson solution for 1 minute, making sure that the volume used is enough to cover the tissue section on each slide completely. Next, rapidly drop 95% ethanol onto the tissue slices for a few seconds to differentiate each tissue section well.
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