Masson Goldner Trichrome Staining: A Technique to Visualize Collagen in Heart Sections for Cardiovascular Fibrosis Detection

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.