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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>

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

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.

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

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.

picrosirius-red-polarization-staining-technique-to-detect-fibrosis

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Biological Techniques

Staining Techniques

Biomolecule staining

1.4K Views. Fonte: Ku, H. C. et al., Un modello di rimodellamento cardiaco attraverso la costrizione dell'aorta addominale nei ratti. J. Vis. Exp. (2016). Questo video descrive la colorazione con rosso picrosirius dei tessuti fibrotici per rilevare l'eccessiva deposizione di collagene nelle sezioni di tessuto fibrotico. La tecnica di colorazione aiuta a valutare l'espressione del collagene nelle malattie del tessuto connettivo. 

Video: Colorazione con polarizzazione rossa Picrosirius: una tecnica per rilevare la fibrosi nelle sezioni di tessuto cardiaco utilizzando la microscopia a campo chiaro - Concept

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression of a wide range of diseases including breast, ovarian, kidney, and pancreatic cancers. Freely available fiber quantification software tools are mainly focused on the calculation of fiber alignment or orientation, and they are subject to limitations such as the requirement of manual steps, inaccuracy in detection of the fiber edge in noisy background, or lack of localized feature characterization. The collagen fiber quantitation tool described in this protocol is characterized by using an optimal multiscale image representation enabled by curvelet transform (CT). This algorithmic approach allows for the removal of noise from fibrillar collagen images and the enhancement of fiber edges to provide location and orientation information directly from a fiber, rather than using the indirect pixel-wise or window-wise information obtained from other tools. This CT-based framework contains two separate, but linked, packages named &#8220;CT-FIRE&#8221; and &#8220;CurveAlign&#8221; that can quantify fiber organization on a global, region of interest (ROI), or individual fiber basis. This quantification framework has been developed for more than ten years and has now evolved into a comprehensive and user-driven collagen quantification platform. Using this platform, one can measure up to about thirty fiber features including individual fiber properties such as length, angle, width, and straightness, as well as bulk measurements such as density and alignment. Additionally, the user can measure fiber angle relative to manually or automatically segmented boundaries. This platform also provides several additional modules including ones for ROI analysis, automatic boundary creation, and post-processing. Using this platform does not require prior experience of programming or image processing, and it can handle large datasets including hundreds or thousands of images, enabling efficient quantification of collagen fiber organization for biological or biomedical applications.

Here, we present a protocol to use a curvelet transform-based, open-source MATLAB software tool for quantifying fibrillar collagen organization in the extracellular matrix of both normal and diseased tissues. This tool can be applied to images with collagen fibers or other types of line-like structures.

Quantificazione dell'organizzazione del collagene fibrilla con strumenti a base di trasformata curvelet

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression ...

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Bioingegneria

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

Infezione da Salmonella cronica Fibrosi intestinale indotta

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...

Immunologia e infezioni

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Isolamento e caratterizzazione di fibroblasti cardiaci adulti e miofibroblasti

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

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

Trascrizione

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