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

Abstract

Source: Valentin, J., et al. Histological Quantification of Chronic Myocardial Infarct in Rats. J. Vis. Exp. (2016). 

This video demonstrates the differential staining of collagen fibers, nuclei, and cytoplasm in heart tissue to visualize fibrotic growth associated with cardiac pathologies.

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

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