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

Protocol

1. Masson-Goldner Trichrome Staining

1. Make tissue sections from paraffin-embedded heart blocks with a manual microtome (thickness: 5 µm).
2. Stain one slide from each heart part for each rat (3 - 4 transversal sections per slide) with Masson-Goldner trichrome staining.
   1. Melt the slides at 60 °C in an oven. Under a fume hood, deparaffinize them in xylol twice for 10 min each. Rehydrate them in 100% ethanol, 95% ethanol, 70% ethanol, and distilled water for 3 min each.
   2. Fix them in Bouin solution overnight. Then, rinse them in running tap water for 10 - 15 min. Rinse them in distilled water. Incubate them in Mayer's hematoxylin for 3 min.
   3. Remove the slides and leave them in distilled water for 5 min. Then, incubate them in acid Fuchsin-Ponceau for 5 min. Rinse them in 1% acetic acid for 1 min.
   4. Next, incubate them in phosphomolybdic acid Orange G for 1 min. Rinse them in 1% acetic acid for 1 min, incubate them in light green dye for 10 min, and rinse them in 1% acetic acid for 1 min.
   5. Dehydrate them in 70% ethanol (30 sec), 95% ethanol (30 sec), and 100% ethanol (5 min). Put one drop of a resinous mounting medium on the tissue sections, cover them with coverslips, and let them dry.

Materials

Name	Company	Catalog Number	Comments
Mayer hematoxylin	MHS32-1L	Sigma Aldrich
Acid Fuchsin CI 42685	F8129-50G	Sigma Aldrich
Ponceau Xylidin CI 16150	P2395-25G	Sigma Aldrich
Orange G CI 16230	O3756-100G	Sigma Aldrich
Light green CI 42095	L5382-25G	Sigma Aldrich
Xylol	10315083	HoneyWell
Ethanol absolute	10303990	HoneyWell
Sterelogical microscope	SM2800	Nikon
Formaldehyde	99340	Reactolab
Embedding cassette	K113.1	Carl Roth
Bersoft Image measurement Software		Bersoft.com	Licensed software