This article discusses a method for visualizing fibrosis in cardiac tissue, specifically focusing on the excessive collagen deposition by stress-activated cardiac fibroblasts. The procedure involves several staining techniques to highlight fibrotic regions under a microscope.
Stress-activated cardiac fibroblasts deposit excessive collagen in injured regions, a condition called fibrosis, which impairs heart function.
To visualize the extent of fibrosis, first, section paraffin-embedded heart blocks. Place the sections on a slide. Heat the slide to melt the paraffin, allowing sections to adhere.
Dip the slide in xylol to deparaffinize the tissue. Treat the sections with descending alcohol strengths, to rehydrate the sections, and aid subsequent binding of aqueous stain components.
Incubate the sections in an acidic fixative that cross-links proteins, stabilizing the tissue structure. Rinse with water to remove residual fixative.
Immerse the slide in hematoxylin solution. The cationic dye binds the negatively-charged DNA, staining the nucleus blue. Rinse with water to remove excess stain.
Incubate the slide in acid Fuchsin-Ponceau, a small-molecule anionic dye that interacts with positively-charged cytoplasmic proteins and collagen, staining them red. Treat with diluted acetic acid, to remove excess stain.
Immerse the slide in phosphomolybdic acid-Orange G solution. Orange G, a small-molecule dye, stains erythrocytes orange-red. Phosphomolybdic acid, a large-molecule acid, expels small-molecule dyes from collagen, rendering it colorless.
Incubate the sections in light-green dye, whose molecules bind collagen fibers, coloring them green. Under a microscope, the fibrotic regions appear green indicating excessive collagen deposition.
In this procedure, prepare five-micron thick tissue sections from the paraffin blocks with a manual microtome. Next, stain one slide from each heart section, for each rat with Masson-Goldner Trichrome staining. For the paraffin sections, melt the tissues at 60 degrees Celsius in an oven. Under a fume hood, de-paraffinize them in xylol twice, for 10 minutes each.
Then, rehydrate them in 100% ethanol, 95% ethanol, 70% ethanol, and then, distilled water for three minutes each. For the de-paraffinized sections, and for the cryo-sections, fix them in Bouin solution overnight. The following morning, rinse them in running tap water for 10 to 15 minutes. Then, rinse again using distilled water.
Fixation in Bouin is the most important step to ensure a splendid Goldner staining.
Incubate them in Mayer's hematoxylin for three minutes. After 3 minutes, remove the slides, and leave them in distilled water for five minutes. Subsequently, incubate them in Ponceau-acid Fuchsin for five minutes, before rinsing with 1% acetic acid for one minute.
Next, incubate the sections in phosphomolybdic acid Orange G for one minute, and then rinse them in 1% acetic acid for one minute. Incubate in light green dye for 10 minutes, and then, rinse in 1% acetic acid for one minute. Finally, dehydrate the sections in 70% ethanol for 30 seconds, 95% ethanol for 30 seconds, and 100% ethanol for five minutes sequentially. Apply one drop of resinous mounting medium onto the tissue sections, cover them with coverslips, and place them to dry.
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