Picrosirius red (PSR) staining is widely used in histology to visualize, study, and quantify collagen fibers, a major component of the extracellular matrix (ECM)¹.

Picrosirius red staining was developed by Dr. Luiz Carlos U. Junqueira in 1979. It is a cost-effective and highly specific method for studying collagen organization in connective tissue pathologies and fibrosis research. It combines Sirius red, a sulfonated azo dye that binds collagen, with picric acid, which enhances specificity and polarization intensity¹.

Under polarized light microscopy, PSR staining distinguishes collagen types I and III, leveraging birefringence to reveal fiber distribution, density, and organization. Collagen bundles appear in distinct hues (green, red, or yellow) against a black background, enabling quantitative morphometric analysis. The addition of picric acid prevents non-specific binding, ensuring clearer differentiation of collagen fibers and improving diagnostic accuracy¹.

Applications of picrosirius red staining

Assessment of collagen organization in tendons: PSR staining, combined with polarized light microscopy, is employed to evaluate the organization of collagen fibers in tendon tissues. This method allows for both qualitative and quantitative assessments, providing insights into the structural arrangement of collagen.

For instance, a study shows that PSR-polarization is useful for visualizing collagen organization, but cannot distinguish between collagen types I and III in tendon tissue. For accurate collagen typing, PSR should be combined with immunohistochemical labeling, and standardized imaging techniques are essential for reliable results².

Visualization of connective tissue in thick sections: Modified PSR staining protocols have been developed to facilitate the localization of connective tissue in thick tissue sections. When analyzed using confocal microscopy, this approach enables detailed three-dimensional visualization of collagen structures.

For example, a fluorescence-based PSR protocol, fully multiplexed with immunostaining, can be imaged by confocal microscopes. This approach treats PSR-stained collagen as a red fluorescent signal, which was shown to be as sensitive as polarized light or second-harmonic generation (SHG) imaging, while being unaffected by section orientation³.

Using laser-scanning confocal z-stacks, one can visualize collagen fiber networks in thick, cleared tissue sections, reconstructing them in 3D. For instance, a study demonstrated confocal imaging of >100 µm thick cleared organ sections with histochemical stains, achieving sub-micron resolution deep into the tissue⁴.

Evaluation of collagen deposition in fibrosis research: In studies investigating fibrosis, PSR staining is utilized to quantify collagen deposition. This technique aids in assessing the extent of fibrotic changes by highlighting collagen accumulation within tissues⁵.

For instance, in a study examining age-associated fibrosis in mouse ovaries, PSR staining combined with confocal microscopy revealed detailed 3D structures of collagen fibers. This approach highlighted the progression of fibrotic changes with age, demonstrating the utility of this method in reproductive biology research⁶.

Analysis of collagen in engineered scaffolds: PSR staining is applied to confirm the presence and distribution of collagen within electrospun extracellular matrix scaffolds. This application is particularly relevant in tissue engineering, where scaffold composition and structure are critical.

For instance, a study functionalized polycaprolactone (PCL) electrospun scaffolds with collagen and osteogenic extracellular vesicles and monitored new collagen deposition using PSR staining. After seeding osteoblast-like cells onto these scaffolds, PSR staining revealed that collagen-coated scaffolds (with or without EVs) developed intense birefringent collagen fibers over 2–3 weeks, whereas uncoated PCL had minimal collagen signal⁷.

Distinction between bone formation and fibrotic tissue in osteosarcoma: In the context of osteosarcoma diagnosis, PSR staining aids in differentiating between bone formation tissues and fibrotic tissue. This distinction is crucial for accurate histopathological evaluation.

For instance, a study investigated Wnt signaling in c-Fos–induced osteosarcomas; PSR with polarized light was used to compare collagen organization in osteoblastic (bone-forming) vs. fibroblastic tumor areas. Tumors with high osteogenic activity showed densely packed, bright red-orange collagen bundles (indicative of thick, type I–rich osteoid matrix) under polarized light, whereas tumors with a more fibrous phenotype showed predominantly thin, loosely packed greenish fibers⁸.

Materials and equipment for staining

The reagents, controls and accessories required for picrosirius red staining may differ according to the experimental conditions⁹. The picrosirius red solution can either be freshly made in a laboratory, or commercially available picrosirius red solutions or assay kits can be used. The amount of dyes used and the incubation time needed can differ for different experiments. Below is the list of reagents needed for the method:

PSR solution

• 0.5 g Sirius red F3BA
• 500 mL saturated aqueous solution of picric acid (1.3% in water)

Acidified water

• 5 mL glacial acetic acid
• 1 L distilled or tap water

Haematoxylin stain: For nuclear staining as part of the protocol⁹.

To perform picrosirius staining effectively, several essential pieces of equipment and materials are needed. The quality of results depends on the proper use of equipment.

Microscope: A standard light microscope or a polarized light microscope can be used to visualize the stained samples. The microscope should have adjustable magnification, polarizing filters and good resolution. Polarized microscopes are preferable for qualitative as well as quantitative analysis of collagen fibers².

Slides and cover slips: Slides and cover slips are used to mount tissue sections for staining and observation. Clean and high-quality glass slides should be used².

Tissue processing equipment: A microtome is used to cut thin tissue sections (4–6 µm) for uniform staining and optimal visualization, while a water bath helps float and flatten these sections before placing them on slides. A paraffin oven is used to embed and prepare tissue sections for cutting¹⁰.

Safety precautions and handling of chemicals

The use of dry picric acid in the laboratory for the preparation of PSR can be hazardous as it is sensitive to heat and friction. Hence, pre-prepared PSR solutions or assay kits can be useful here¹¹.

Although commercial assay kits do not contain hazardous chemicals, it is important to take precautions to ensure safety and protect the environment.

• Ensure proper precautionary measures by wearing protective gloves, eye protection, and face protection.
• Maintain good industrial hygiene practices.
• Use adequate ventilation systems.
• Ensure access to showers and eyewash stations.
• Use respiratory protection (national institute for occupational safety and health (NIOSH)/ mine safety and health administration (MSHA) approved) if exposure limits are exceeded or irritation occurs¹².
• Positive pressure supplied by air respirators may be required for high airborne contaminant concentrations.
• Follow local regulations for respiratory protection.

Picrosirius red staining protocol

The following protocol should be employed for the picrosirius red staining:

Preparation: Ensure all materials and reagents are at room temperature and mix gently before use.

Deparaffinization: Deparaffinize the tissue sections if needed and rehydrate them using distilled water.

Staining: Cover the tissue section completely with picrosirius red solution and let it incubate for 60 minutes.

Rinsing: Briefly rinse the slide in two changes of acetic acid solution, then in absolute alcohol.

Dehydration: Dehydrate the tissue section with two changes of absolute alcohol.

Clearing and mounting: Clear the slide and mount it using synthetic resin.

Assay kits like picrosirius red stain kit (connective tissue stain) can be used to see collagen I and III fibers, and picrosirius red stain kit (cardiac muscle) can be used to visualize thin septa and collagen fibers.

Considerations for optimal results, especially for polarized light microscopy

Avoid over-rinsing or prolonged washing steps, as this may reduce staining intensity.

Uniformly cut tissue sections at an optimal thickness (eg, 4-6 μm) to ensure consistent staining and polarization effects¹⁰.

Ensure proper alignment of the polarizer and analyzer in the microscope for clear birefringence patterns when using polarized light².

The sub-stage condenser height and field iris diaphragm should be appropriately aligned for enhanced illumination².

Specific conditions for collagen detection and polarization effects

Picrosirius red binds explicitly to collagen fibers, making them highly visible under standard or polarized light microscopy. Polarized light reveals collagen fiber birefringence, where thick fibers appear yellow, orange, or red, and thin fibers show green or greenish-yellow hues. Optimal polarization effects require staining and tissue samples with significant collagen content².

Visualization and interpretation

Picrosirius red staining can be viewed with standard light microscopy or polarized light. Polarized light enhances the differentiation of collagen fibers from the background and can reveal variations in fiber appearance. However, these differences may result from collagen fiber orientation, thickness, or packing rather than indicating distinct collagen types.

Microscopy techniques

Tissues can be easily visualized under microscopes to study their alignment and architecture. For this purpose, two types of microscopy techniques can be used namely – bright-field microscopy and polarized light microscopy.

Bright-field light microscopy

Bright-field microscopy is a standard technique used to visualize stained tissue sections. Picrosirius red staining enhances the visibility of collagen fibers under bright-field microscopy, allowing for the qualitative assessment of collagen distribution in tissues¹³.

While bright-field microscopy can show the presence of collagen, it lacks the sensitivity to differentiate collagen fiber types (eg, thin vs. thick fibers). All types of collagens appear in a single color. Additionally, the structure of the fibers may not be as clearly defined compared to polarized light microscopy. The colors of the different parts are:

Cytoplasm and muscles look yellow

All types of collagens appear to be red

Polarized light microscopy

Tissue sections are analyzed using polarized light microscopy with a microscope equipped with a digital camera and a rotating polarizer for optimal imaging. This helps with quantitative collagen analysis.

The exposure time is adjusted to capture the brightest birefringence of collagen fibers or other structures without pixel saturation, and the stage is motorized to allow for precise sample rotation. To assess the effect of tissue orientation, the slide holder is rotated in increments, capturing images at different angles, with images stitched to cover the entire cross-sectional area for consistent analysis².

Birefringence and its role in collagen detection

Birefringence, an optical property in collagen, reflects changes in fiber organization and structure during processes like fibrosis. As collagen fibers mature, increased cross-linking, dehydration, and proteoglycan changes enhance birefringence intensity and shift polarization colors from green (immature fibers) to orange or red (mature fibers)¹¹. This property is used for:

• Collagen cross-linking detection.
• Evaluating the organization and alignment.
• Understanding degradation of collagen in pathological conditions leading to loss of polarization sensitivity and structural disorganization¹¹.

Digital image analysis for quantitative assessment

Digital image analysis (DIA) techniques for collagen quantification evolved from grayscale to color imaging to improve accuracy, including background subtraction and hue-based fiber thickness assessments.

However, user-adjustable parameters in some methods can introduce variability, while grayscale analysis, as used in this study, avoids such issues and focuses on assessing overall fibrillar collagen without separating by color or thickness.

Future studies could enhance these methods by comparing circular and linear polarization imaging techniques against gold standards like collagen-specific assays to better evaluate collagen structure and concentration¹⁴.

Digital image analysis is performed by using software to quantify total collagen and background based on specific threshold settings for the red, green, and blue channels. Background thresholds are set at lower values for each channel. The software automatically calculates pixel areas, allowing for the determination of the percentage of collagen and background in each tissue².

Interpreting results

Identifying collagen networks, types, and organization under different lighting: Collagen visualization with picrosirius staining highlights collagen fibers as birefringent structures, allowing their clear image against the surrounding tissue. Collagen networks can be assessed for their density, distribution, and alignment. It can also identify the wound healing process and tissue regeneration process. The color variations and birefringence patterns allow researchers to distinguish between collagen types and assess their arrangement.

Histological collagen visualization can be achieved using various techniques, each with its strengths and limitations. High-resolution methods like electron microscopy and second harmonic generation imaging provide detailed collagen visualization but are costly and complex, while PSR staining offers an affordable, reproducible, and widely used method that enhances collagen birefringence under polarized light³.

Fluorescent imaging of PSR-stained samples avoids orientation-based limitations of linear polarized light, producing a strong, collagen-specific red fluorescence signal, though circularly polarized light also provides improved consistency but requires specialized equipment.

Brightfield light displays collagen in red tones against a light yellow or colorless background. Polarized light enhances birefringence and displays collagen in vibrant colors, allowing a more detailed analysis of fiber types and maturation².

This ability to switch between lighting modes helps researchers identify both the quantity and structural integrity of collagen, making it particularly useful for assessing tissue remodeling or pathological changes. For example:

Collagen I: These are thick fibers found in the bones, tendons and late stage wounds, forming parallel bundles essential for resisting mechanical forces. It appears orange red under a polarized microscope².

Collagen III: These are fibers found in the skin and connective tissue and form a meshwork involved in tissue regeneration and early wound healing. It appears green under a polarized microscope².

Collagen IV: Found in the basement membranes. But as it is non-fibrillar, it cannot be seen under a PSR stain polarized light microscope¹⁵.

After tissue injury, collagen type III content often increases, particularly in conditions like Achilles tendon rupture. Such imbalances in collagen composition are also observed through polarized PSR staining to assess tissue damage in the body².

Troubleshooting and optimization

Even with strict adherence to protocols and precautions, challenges may still arise during staining procedures. Fortunately, these issues can often be resolved with targeted optimization strategies. Below is a detailed list of potential problems and their corresponding solutions to ensure consistent and accurate staining results.

Dry tissue sections

• Problem: Dried sections show a higher background at the edges.
• Solution: Keep tissue sections in a humidified chamber to prevent drying.

Inadequate washing

• Problem: Residual fixatives or unbound antibodies cause false positives.
• Solution: Extend washing time and ensure thorough rinsing between steps.

Inadequate deparaffinization

• Problem: Insufficient removal of paraffin in sections.
• Solution: Extend deparaffinization time using fresh xylene.

Damaged cell membranes

• Problem: Permeabilization affects membrane integrity.
• Solution: Use gentler detergents like tween 20 instead of triton X and remove permeabilizing agents from buffers if unnecessary.

Improper storage of reagents

• Problem: Antibodies lose activity due to improper handling.
• Solution: Follow storage instructions on datasheets and avoid repeated freeze-thaw cycles.

Using freshly prepared reagents, increasing washing steps and maintaining standardizing conditions can optimize the staining process effectively.

Advanced techniques and future directions

Recent advances in digital pathology, such as curvelet transform-fiber extraction (CT-FIRE) software analysis of PSR stains under polarized light, have enabled the identification of collagen fiber descriptors as diagnostic and prognostic biomarkers in lung cancer.

This approach revealed that collagen straightness is a high-accuracy diagnostic marker, while fiber density correlates with poor prognosis. This shows the potential of collagen analysis in clinical settings and unveils a stiff tumor microenvironment in lung adenocarcinoma that promotes immune evasion and tumor progression, suggesting promising avenues for therapeutic research and regenerative medicine.

Additionally, these advancements are helping researchers explore the relationship between collagen organization and disease progression, such as in cancer, thus opening new possibilities for diagnostics and therapeutic interventions across various fields¹⁶.

FAQs

What is the difference between Sirius red and picrosirius red staining?

The main difference between Sirius red and picrosirius red staining lies in the composition of the staining solution. Sirius red staining can only differentiate the collagen from non-collagen areas. However, in some cases, it may lead to some non-specific bindings. Picrosirius red removes the non-specific binding and provides a detailed visualization of different collagen types. Thus, Picrosirius red staining is often preferred in research as it provides stronger birefringence under polarized light, making it more suitable for analyzing collagen structure and organization.

Can picrosirius red staining differentiate between different types of collagens?

Yes, picrosirius red staining can differentiate between various types of collagens, particularly type I and type III. Under polarized light, type I collagen fibers typically appear red or orange, while type III collagen fibers exhibit a green or yellow hue. This differential staining helps researchers assess the collagen composition and organization in tissues, making PSR staining valuable for studying fibrosis, tissue remodeling, and various diseases.

What is the protocol for tissue section preparation for Sirius red staining?

For Sirius red staining, tissue samples are first fixed in cold 4% neutral formalin and processed for paraffin embedding. Thin sections (3-5 µm) are cut, deparaffinized, and rehydrated before being incubated with a 0.1% Sirius red solution in aqueous picric acid for 1 hour. After staining, the slides are washed, dehydrated, and mounted with a resin medium for visualization under a microscope.

Is picrosirius red staining quantitative?

Yes, picrosirius red (PSR) staining can be quantitative when combined with image analysis techniques. By using polarized light microscopy or digital imaging, researchers can analyze the intensity and distribution of collagen fibers in tissue sections, providing quantifiable data on collagen content, fiber density, and organization. Advanced software tools can be used to assess these parameters, enabling researchers to correlate collagen deposition with disease progression, tissue remodeling, and therapeutic outcomes.

References

1. Lattouf, R., Younes, R., Lutomski, D., et al. Picrosirius Red Staining. Journal of Histochemistry & Cytochemistry. 62 (10), 751–758 (2014).
2. López De Padilla, C. M., Coenen, M. J., Tovar, A., et al. Picrosirius Red staining: revisiting its application to the qualitative and quantitative assessment of collagen type I and type III in tendon. Journal of Histochemistry & Cytochemistry. 69(10), 633–643 (2021)
3. Wegner, K. A., Keikhosravi, A., Eliceiri, K. W., et al. Fluorescence of Picrosirius Red multiplexed with immunohistochemistry for the quantitative assessment of collagen in tissue sections. Journal of Histochemistry & Cytochemistry. 65(8), 479–490 (2017)
4. High-resolution 3D fluorescent imaging of intact tissues. International Journal of Cardiology and Cardiovascular Diseases. 1(1) (2021).
5. Segnani, C., Ippolito, C., Antonioli, L., et al. Histochemical detection of collagen fibers by Sirius Red/Fast Green is more sensitive than van Gieson or Sirius Red alone in normal and inflamed rat colon. PLOS ONE. 10(12), e0144630 (2015).
6. Briley, S. M., Jasti, S., McCracken, J. M., et al. Reproductive age-associated fibrosis in the stroma of the mammalian ovary. Reproduction. 152(3), 245–260 (2016).
7. Nieuwoudt, M., Woods, I., Eichholz, K. F., et al. Functionalization of electrospun polycaprolactone scaffolds with matrix-binding osteocyte-derived extracellular vesicles promotes osteoblastic differentiation and mineralization. Annals of Biomedical Engineering. 49(12), 3621–3635 (2021).
8. Matsuoka, K., Bakiri, L., Wolff, L. I., et al. Wnt signaling and Loxl2 promote aggressive osteosarcoma. Cell Research. 30(10), 885–901 (2020).
9. Stephenson, B. A modified Picro-Sirius Red (PSR) staining procedure with polarization microscopy for identifying collagen in archaeological residues. Journal of Archaeological Science. 61, 235–243 (2015).
10. Cooper, C., Thompson, R. C. A., Clode, P. L. Investigating parasites in three dimensions: trends in volume microscopy. Trends in Parasitology. 39(8), 668–681 (2023).
11. Gopinathan, P. A., Kokila, G., Jyothi, M., et al. Study of collagen birefringence in different grades of oral squamous cell carcinoma using Picrosirius Red and polarized light microscopy. Scientifica. 2015, 1–7 (2015).
12. Derivation of Immediately Dangerous to Life or Health (IDLH) Values. CURRENT INTELLIGENCE BULLETIN 66, DEPARTMENT OF HEALTH AND HUMAN SERVICES, Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health.
13. Gaytan, F., Morales, C., Reymundo, C., et al. A novel RGB-trichrome staining method for routine histological analysis of musculoskeletal tissues. Scientific Reports. 10(1) (2020).
14. Greiner, C., Grainger, S., Farrow, S., et al. Robust quantitative assessment of collagen fibers with Picrosirius Red stain and linearly polarized light as demonstrated on atherosclerotic plaque samples. PLOS ONE. 16(3), e0248068 (2021).
15. Lopera Higuita, M., Shortreed, N. A., Dasari, S., et al. Basement membrane of tissue-engineered extracellular matrix scaffolds modulates rapid human endothelial cell recellularization and promotes quiescent behavior after monolayer formation. Frontiers in Bioengineering and Biotechnology. 10 (2022)
16. Almici, E., Arshakyan, M., Carrasco, J. L., et al. Quantitative image analysis of fibrillar collagens reveals novel diagnostic and prognostic biomarkers and histotype-dependent aberrant mechanobiology in lung cancer. Modern Pathology: An Official Journal of the United States and Canadian Academy of Pathology, Inc. 36(7), 100155 (2023).