Picrosirius Red Polarization Staining: A Technique To Detect Fibrosis in Cardiac Tissue Sections Using Bright-Field Microscopy

Overview

This video describes the picrosirius red staining of fibrotic tissues to detect excessive collagen deposition in fibrotic tissue sections. The staining technique helps assess collagen expression in connective-tissue diseases.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Tissue Fibrosis Quantification

1. Place the paraformaldehyde-fixed heart tissue on a tissue sectioning device and cut 2 mm thick sections. Place the tissue sections in an embedding cassette. Dehydrate the tissue through a series of graded alcohol baths (50%, 75%, 95%, and 100% for 1 hr each).
2. Infiltrate the tissue with xylene for 1 hr and finally in wax for 1 hr. Place the infiltrated tissue into an embedding cassette and embed with paraffin wax. Store the embedded tissues in paraffin blocks at room temperature until microtoming.
3. Slice the embedded tissue into 4 μm thick sections. Place the sections in a 45 ºC water bath. Dip a glass slide into the water bath at an angle and gradually approach the paraffin section edges to allow partial attachment to the slide.
4. Move the slide in and out of the bath to remove potential air pockets under the tissue section and to facilitate better attachment. Dry the slides at 37 ºC for 1 hr and store them at room temperature for histological staining.
5. Put the slide into a tank. Deparaffinize the slide with xylene for 30 min and rehydrate it in sequentially diluted alcohol (95%, 75%, and 50% for 3 min each) and finally in distilled water. Use adequate picrosirius red solution to completely cover the tissue sections for 1 hr. Rinse the slides in a 0.5% acetic acid solution for two changes, and then perform two rinses in absolute alcohol.
6. Air-dry the slide and mount the slide in synthetic resin with a coverslip. Photograph the slide in a visible light field.
   NOTE: The red area in the photograph shows a picrosirius red positive zone under a microscope at 200X magnification. Calculate the percentage of the picrosirius red positive zone over the total area, which indicates the extent of fibrosis.

Materials

Name	Company	Catalog Number	Comments
Paraformaldehyde	Sigma Aldrich	441244
Picrosirius red solution	Abcam	ab150681
Imagequant	Molecular Dynamics