picrosirius red polarization staining: a technique to detect fibrosis in cardiac tissue sections using bright-field microscopy

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

After fixing the heart tissue, place it on a tissue sectioning device, cut it into 2-millimeter-thick slices, and place the sections in an embedding cassette.
Following paraffin embedding and additional sectioning according to the text protocol, deparaffinize the slides by placing them into a tank with xylene for 30 minutes. Then, rehydrate the tissue in a sequentially diluted alcohol series, followed by distilled water.
Next, use adequate picrosirius red solution to completely cover the tissue sections, and incubate for one hour. Then, use a 0.5% acetic acid solution to rinse the slides two times, and rinse the samples twice in absolute alcohol.
Air-dry the samples, and mount them in synthetic resin with a coverslip. Then, use visible light under a microscope at 200X magnification to image the slides.

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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
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>Slice the embedded tissue into 4 &#956;m thick sections. Place the sections in a 45 &#186;C 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.</li>
	<li>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 &#186;C for 1 hr and store them at room temperature for histological staining.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

<table><tbody><tr><td>Paraformaldehyde&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>441244</td><td></td></tr><tr><td>Picrosirius red solution&amp;nbsp;</td><td>Abcam&amp;nbsp;</td><td>ab150681</td><td></td></tr><tr><td>Imagequant&amp;nbsp;</td><td>Molecular Dynamics</td><td></td><td></td></tr></tbody></table>

Source: Ku, H. C. et al., <a href="https://www.jove.com/v/54818">A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats.</a> J. Vis. Exp. (2016).&#160;
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.

Source: Ku, H. C. et al., A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats. J. Vis. Exp. (2016).&#160;
This video ...

Fibrosis is an increased deposition of fibrillar collagen in the extracellular matrix of the damaged tissue. Structurally, it comprises triple-helix collagen polypeptide chains.
To stain collagen with picrosirius red dye, take a paraformaldehyde-fixed, paraffin-embedded, cardiac tissue section. Immerse the tissue section in xylene - an organic solvent - to dissolve the surrounding wax.
Sequentially treat the deparaffinized tissue with decreasing concentrations of alcohol to replace the xylene. Submerge the tissue in water to rehydrate it for better stain visualization in the subsequent steps.
Dip the tissue into picrosirius red solution containing Sirius red - an anionic azo dye - and picric acid.
Sirius red contains acidic sulfonic groups that interact with the basic amino groups of lysine, hydroxylysine, and arginine present in the collagen. The picric acid helps maintain the acidic pH, facilitating the binding of dye molecules in a parallel orientation with the collagen.
Rinse the slides in an acidified destaining solution to remove excess stain. Put a drop of mounting medium on the slide and view it under a bright-field microscope.
The collagen-containing fibrotic area appears bright red compared to the non-fibrotic, yellow-colored regions.

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

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