Method Article

Picrosirius Red Staining for Semiquantitative Histopathologic Evaluation of Collagen Deposition in Murine Models of Chronic Lung Allograft Rejection

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DOI:

10.3791/66395

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March 21st, 2025

 ,  ,  ,  ,  ,  ,  ,  , 
1Department of Thoracic Surgery, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, 2Department of Plastic and Hand Surgery, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, 3Department of Thoracic Surgery, Kyoto University Hospital, 4Department of Otolaryngology, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg

* These authors contributed equally

In This Article

Summary

Picrosirius Red staining is a semiquantitative method for objectively assessing collagen deposition in murine pulmonary fibrotic remodeling in a spatially resolved manner. Apart from evaluating total collagen deposition, Picrosirius Red staining allows for differentiating collagen fibers of different thicknesses.

Abstract

Fibrosis is the pathophysiologic hallmark of chronic rejection after lung transplantation and the foremost hurdle to long-term recipient survival. Several murine lung transplantation models are available for the study of chronic rejection. However, they display heterogeneous results regarding fibrotic changes of the graft, and the histologic extent of fibrosis is mostly reported qualitatively. Therefore, a spatially resolved approach that allows for statistical analysis can aid in evaluating fibrosis in these models. This study presents Picrosirius Red staining for a semiquantitative evaluation of collagen organization in murine lung allografts and compares it to standard Hematoxylin and Eosin, Masson's Trichrome, and Herovici's stains. Staining was performed on sections from two different murine transplantation models based on minor and major histocompatibility complex (MHC) mismatches. The method was established for semiquantitative analysis of collagen organization in whole lung sections. Thus, it can serve as a tool for murine experimental models of fibrotic lung diseases.

Introduction

Lung transplantation is the definite therapeutic option for patients suffering from end-stage lung disease. However, long-term survival is hampered by chronic rejection, affecting 50% of recipients within the first five postoperative years1. Fibrotic remodeling of the small airways and the lung parenchyma is the histologic hallmark underlying the progressive loss of pulmonary function in chronic lung allograft rejection2. Experimentally, chronic lung allograft rejection can be modeled with murine orthotopic lung transplantation between MHC-mismatched mouse strains. Different strain combinations have been proposed to achieve the phenotype of chronic rejection3. Among them, the transplant combination of the minor MHC mismatched C57BL/10 donor and C57BL/6 recipient is frequently used4. Alternatively, BALB/c donor lungs can be transplanted to C57BL/6 recipients receiving daily immunosuppressive treatment5. These models result in histologically different degrees of fibrotic changes6. These changes are often reported qualitatively using the standard Hematoxylin and Eosin, and Masson's Trichrome staining.

The standard histological staining with Hematoxylin and Eosin allows for an overview of the specimen as it stains nuclei blue, while cytoplasm and collagen fibers appear red. With Masson's Trichrome staining, nuclei are stained black, collagen fibers are stained green to blue, and the background, including cytoplasm, fibrin, and muscles, are red. Because Masson's Trichrome stain may lead to underestimated values, using Picrosirius Red stain is considered to be beneficial7. Picrosirius Red is a linear anionic dye that associates with long cationic collagen fibers and provides contrast-rich red staining. Furthermore, Picrosirius Red staining enhances the natural birefringence of collagen fibers under cross-polarized light8. This way, collagen fiber thickness and packing can be evaluated. Using a polarizing filter, the background turns dark, and distinguishing the thickness of deposited collagen becomes possible. While thick collagen fibers appear red, thin collagen fibers emerge green under polarization. A direct association between birefringence and collagen subtype is frequently described in the literature, with red birefringence assigned to collagen I and green birefringence to collagen III9.

This protocol describes the use of Picrosirius Red staining to evaluate fibrosis in murine lung allografts. In addition to conventional histological staining, it allows for a semiquantitative evaluation of fibrotic change in murine models of chronic rejection, provides a means of distinction between thick and thin collagen fibers in only one staining, is cost-efficient, and is easy to perform. This method can equally be applied to other murine experimental models characterized by fibrotic remodeling of the lung parenchyma.

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Protocol

All animal protocols comply with the ethical principles of the 3Rs for humane animal research and were approved by the local veterinary ethical committee (Veterinäramt Kanton Zürich, Switzerland, Study number 45/2014). Likewise, readers should obtain permission from the relevant institutions before performing any procedures on laboratory animals.

1. Sample acquisition

  1. Conduct animal experiments according to individual needs and respective ethical approval. A single microsurgeon performs all surgeries without any additional prolonged ischemia times.
    NOTE: For a reproduction of our representative results, obtain specific pathogen-free adult male C57BL/6J, C57BL/10J, and BALB/c mice weighing 27-30 g from Charles River Laboratories. For isografts, use C57BL/6J animals as both donors and recipients. In the minor mismatched model, transplant C57BL/10J donor lungs to C57BL/6J recipients. In the major mismatched model, transplant BALB/c donor lungs to C57BL/6J recipients and postoperatively inject cyclosporine (10 mg/kg/day) and methylprednisolone (1.6 mg/kg/day) subcutaneously daily5. Use saline solution for perfusion and retrieve lungs after 8 weeks (56 days).
  2. For euthanasia, intubate the animal with a 20G catheter and ventilate it with O2 supplemented with 2%-3% isoflurane. Confirm sufficient depth of anesthesia by the absence of a response to a paw pinch.
  3. Perform a median laparosternotomy by incising the abdominal wall with surgical scissors and cutting the length of the sternum to expose the heart. Identify the inferior vena cava and the left atrial appendage by their anatomical location.
  4. In quick succession, cut the inferior vena cava and the left atrial appendage with surgical scissors and perfuse the animal with 3-5 mL of 0.9% NaCl through the root of the pulmonary artery with a syringe to remove all blood from the pulmonary circulation.
    NOTE: Euthanasia should be performed according to the respective ethical approval. Other methods can be equally applied. It is recommended that blood from the pulmonary circulation be thoroughly removed, which can also be performed by perfusion after circulatory arrest.
  5. Remove the perfused lungs using scissors.
  6. For conservation, incubate the samples in 4% formalin. Inject the formalin solution directly into the bronchus and pulmonary vasculature. Submerge the samples in formalin and let them incubate for at least 6 h.
    NOTE: For inflation of the lung with formalin, a standardized procedure should be applied to obtain comparable results. Example protocols for such procedures are published elsewhere10,11.
  7. Trim the organ according to experimental needs. Transversally cut the lungs in half to capture all parts of the bronchial tree in a section.
  8. Transfer samples to an automatic tissue processor for dehydration in alcohol sequence, clearing in xylol and paraffin coating as follows: 70% ethanol, 70% ethanol, 80% ethanol, 96% ethanol, 100% ethanol (1 h each), 100% ethanol, 100% ethanol, xylol (2 h each), xylol (1 h), and paraffin 3x for 2 h.
  9. Melt paraffin and embed samples in the correct orientation in Polyoxymethylen embedding cassettes. Take care to embed the samples with the cut surface facing downwards. Use a rotary microtome to cut 5 µm sections consecutively. Let the slides dry for 24 h at 37 °C before further processing.

2. Deparaffinization and hydration of paraffin slides

  1. Remove paraffin by incubating the samples 2x in 100% xylol for 5 min each.
    CAUTION: Xylol is flammable and hazardous to health. Only use under the fume hood.
  2. Hydrate the samples in 100% ethanol, 100% ethanol, 95% ethanol, 70% ethanol, and 50% ethanol for 2 min each. Then, wash for 2 min in distilled water.

3. Staining with Picrosirius Red

  1. Stain slides for 8 min in Meyer's hemalum solution. Wash slides for 10 min under running tap water.
  2. Incubate slides for 1 h in Picrosirius Red solution. For Picrosirius Red solution, either use a premixed staining solution or use 0.1% of Direct Red 80 or Sirius Red F 3B (CI 35780) diluted in saturated picric acid.
    CAUTION: Picric acid is flammable and highly explosive and should be treated carefully.
    NOTE: There are many different suppliers for Picrosirius Red staining kits available, as listed in the Table of Materials. Be aware of differences in price and staining methods, and always adhere to suppliers' instructions. It is recommended to only use staining kits with the specific color index for Sirius Red F 3B (CI 35780).
  3. Dip the slides 5x-10x in an acetic acid solution (0.5%). Gently shake the slides to physically remove the solution.
  4. Dehydrate the slides in 95% ethanol, 100% ethanol, 100% ethanol, 100% xylol, and 100% xylol for 5 min each.
  5. Embed by adding one drop of mounting medium on each sample and cover with a coverslip. Let slides dry under a fume hood.

4. Digitalization and image processing

  1. Focus the samples under a light microscope with appropriate imaging software until the picture becomes clear and sharp. Add a polarizing filter and adjust the degree of polarization until the background is completely dark or black. When the background is as dark as possible, the correct polarization is achieved.
    NOTE: It is also possible to visualize Picrosirius Red staining with conventional transmitted light and without a polarizing filter. For this purpose, it is recommended to use Sirius Red with Fast Green, as it has been shown to be more sensitive than Sirius Red alone12,13. However, polarizing microscopy is necessary to distinguish between thick and thin collagen fibers.
  2. Scan all samples completely under 20x magnification and export as .tiff. Transfer to Fiji software14. Make sure to clean slides properly in advance and use standardized settings regarding exposure time and light source intensity.
  3. Surround the sample manually and cut it out in the software using Edit > Clear Outside. Measure the total surface area by clicking Analyze > Measure.
    NOTE: The selection of the compartment to measure is a critical step in creating reliable and comparable results. By digitalizing and selecting the entire sample, a good cross-section is displayed, and heterogeneous tissues are considered as a whole. However, it is also possible to confine oneself to specific areas within the samples, such as pleura or individual bronchi. Keep in mind to always select compartments to compare in a standardized fashion. Should the samples show a large variability in ventilation, it is advisable to exclude the airspace area from the analyzed compartment15.
  4. Measure total collagen content per sample using color threshold by clicking Image > Adjust > Color Threshold and adjust the settings by clicking Analyze > Analyze Particles. Set Size = 1 - Infinity, Clear, Summarize. Brightness = 35 - 255. Hue = 2 - 130
    NOTE: A reasonable setting of the color thresholds is critical to achieve reliable results.
  5. Repeat for thick and thin collagen fibers by adjusting Hue as Red Approximation symbol, mathematical notation, equation representation, educational use. Thick Collagen Fibers (Hue 2-30) and Yellow-Green Approximation symbol, mathematical notation, equation representation, educational use. Thin Collagen Fibers (Hue 31-130). The result will be the area of stained pixels.
  6. Configure Macro for automated processing by clicking Plugins > Macros > Record and repeat with all samples.
  7. To receive final values standardized to the cross-section's surface area, divide the area of stained pixels for all parameters by the total surface area as measured in steps 4.3 and 4.5 for each sample, respectively.

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Results

The protocol described above allows for an objective semiquantitative evaluation of collagen deposition in murine lung tissue. Fibrotic remodeling is the pathophysiological hallmark of chronic rejection after lung transplantation. Therefore, Picrosirius Red staining was applied in chronic lung allograft rejection models using left-sided murine orthotopic lung transplantation. The major MHC-mismatched lung transplantation from a BALB/c donor to a C57BL/6 recipient under mild immunosuppression results in fibrotic changes c...

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Discussion

Standard histologic methods such as Hematoxylin and Eosin, and Masson's Trichrome staining are widely used to detect fibrotic changes in murine lungs in a spatially resolved manner16,19. However, additional methods are often necessary to quantify these changes and evaluate the tissue's collagen composition.

Picrosirius Red staining was first described in 1964 to identify collagen: under transmitted light microscopy, collagen ap...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

Birte Ohm is supported by the Berta-Ottenstein-Program for Clinician Scientists, Faculty of Medicine, University of Freiburg. Steffen U Eisenhardt is a Heisenberg Professor of the German Research Foundation (DFG) and supported this work with personal grants. Furthermore, we would like to thank Sheena Kreuzaler for her technical assistance. Figure 1 was created with the help of Biorender.com. Imaging was performed at the Lighthouse Core Facility, which is funded in part by the Medical Faculty, University of Freiburg (Project Numbers 2023/A2-Fol; 2021/B3-Fol), the DKTK, and the DFG (Project Number 450392965).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Acetic acidHoneywell, Charlotte, USA33209
Axio ObserverZeiss, Oberkochen, Germany4633000956 (serial number)
Coverslip 1.5Roth, Karlsruhe, GermanyKCY5.1
Formaldehyde 37%Fisher Scientific, Leicestershire, UKF/1501/PB15
Meyer's hemalum solutionMerck, Darmstadt, Germany109249
Picrosirius Red SolutionMorphisto, Offenbach am Main, Germany13422alternatives that can be used: ab150681, abcam, Cambridge, UK; SRS250 ScyTek Laboratories, Logan City US
Polarizing filterZeiss, Oberkochen, Germany000000-1121-813
Rotary microtome, HistoCore AUTOCUTLeica, Wetzlar, Germany149AUTO00C1, 14051956472
ROTI Histokitt mounting mediumRoth, Karlsruhe, Germany6638.1
ROTI Plast ParaffinRoth, Karlsruhe, Germany6642.5
Rotilabo-embedding cassettes, POMRoth, Karlsruhe, GermanyK113.1
Superfrost Plus Adhesion Microscope slideepredia, Portsmouth, UKJ1800AMNZ
Tissue Processor Leica, Wetzlar, GermanyTP 1020
Software
Fiji software version 2.14.0/1.54fOpen Source
Imaging Software ZEN 3.4.91Zeiss, Oberkochen, Germany

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Tags

Chronic Lung RejectionMurine Lung TransplantationSemiquantitative EvaluationFibrosis AnalysisPolarization MicroscopyMasson s TrichromeDigital Image Analysis

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