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.&#160;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).

All animal protocols comply with the ethical principles of the 3Rs for humane animal research and were approved by the local veterinary ethical committee (Veterin&#228;ramt Kanton Z&#252;rich, Switzerland, Study number 45/2014). Likewise, readers should obtain permission from the relevant institutions before performing any procedures on laboratory animals.
1. Sample acquisition
<ol>
	<li>Conduct animal experiments according to individual needs and respective ethical approval. A single microsurgeon performs all surgeries without any additional prolonged ischemia times. 
	NOTE:&#160;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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Remove the perfused lungs using scissors.</li>
	<li>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.</li>
	<li>Trim the organ according to experimental needs. Transversally cut the lungs in half to capture all parts of the bronchial tree in a section.</li>
	<li>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.</li>
	<li>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 &#181;m sections consecutively. Let the slides dry for 24 h at 37 &#176;C before further processing.</li>
</ol>
2. Deparaffinization and hydration of paraffin slides
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Staining with Picrosirius Red
<ol>
	<li>Stain slides for 8 min in Meyer&#39;s hemalum solution. Wash slides for 10 min under running tap water.</li>
	<li>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&#39; instructions. It is recommended to only use staining kits with the specific color index for Sirius Red F 3B (CI 35780).</li>
	<li>Dip the slides 5x-10x in an acetic acid solution (0.5%). Gently shake the slides to physically remove the solution.</li>
	<li>Dehydrate the slides in 95% ethanol, 100% ethanol, 100% ethanol, 100% xylol, and 100% xylol for 5 min each.</li>
	<li>Embed by adding one drop of mounting medium on each sample and cover with a coverslip. Let slides dry under a fume hood.</li>
</ol>
4. Digitalization and image processing
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Surround the sample manually and cut it out in the software using Edit &#62; Clear Outside. Measure the total surface area by clicking Analyze &#62; 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.</li>
	<li>Measure total collagen content per sample using color threshold by clicking Image &#62; Adjust &#62; Color Threshold and adjust the settings by clicking Analyze &#62; 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.</li>
	<li>Repeat for thick and thin collagen fibers by adjusting Hue as Red <img alt="Equation 1" src="/files/ftp_upload/66395/66395eq01.jpg" style="margin: auto;" /> Thick Collagen Fibers (Hue 2-30) and Yellow-Green <img alt="Equation 1" src="/files/ftp_upload/66395/66395eq01.jpg" style="margin: auto;" /> Thin Collagen Fibers (Hue 31-130). The result will be the area of stained pixels.</li>
	<li>Configure Macro&#160;for automated processing by clicking Plugins &#62; Macros &#62; Record and repeat with all samples.</li>
	<li>To receive final values standardized to the cross-section&#39;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.</li>
</ol>

Collagen Assessment in Murine Pulmonary Fibrosis Using Picrosirius Red Staining

<ol>
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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+lung+transplant+research:+A+consensus+statement+from+the+National+Heart,+Lung,+and+Blood+Institute+workshop.">Models of lung transplant research: A consensus statement from the National Heart, Lung, and Blood Institute workshop.</a> JCI Insight. 2 (9), e93121 (2017).</li><li>Martinu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrum+of+chronic+lung+allograft+pathology+in+a+mouse+minor-mismatched+orthotopic+lung+transplant+model.">Spectrum of chronic lung allograft pathology in a mouse minor-mismatched orthotopic lung transplant model.</a> Am J Transplant. 19 (1), 247-258 (2019).</li><li>de Vleeschauwer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+rejection+pathology+after+orthotopic+lung+transplantation+in+mice:+The+development+of+a+murine+BOS+model+and+its+drawbacks.">Chronic rejection pathology after orthotopic lung transplantation in mice: The development of a murine BOS model and its drawbacks.</a> PLoS One. 7 (1), e29802 (2012).</li><li>Yamada, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+airway+fibrosis+in+orthotopic+mouse+lung+transplantation+models-An+experimental+reappraisal.">Chronic airway fibrosis in orthotopic mouse lung transplantation models-An experimental reappraisal.</a> Transplantation. 102 (2), e49-e58 (2018).</li><li>Whittaker, P., Kloner, R. A., Boughner, D. R., Pickering, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+myocardial+collagen+with+picrosirius+red+staining+and+circularly+polarized+light.">Quantitative assessment of myocardial collagen with picrosirius red staining and circularly polarized light.</a> Basic Res Cardiol. 89 (5), 397-410 (1994).</li><li>Puchtler, H., Waldrop, F. S., Valentine, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization+microscopic+studies+of+connective+tissue+stained+with+picro-sirius+red+FBA.">Polarization microscopic studies of connective tissue stained with picro-sirius red FBA.</a> Beitr Pathol. 150 (2), 174-187 (1973).</li><li>Junqueira, L. C. U., Cossermelli, W., Brentani, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+staining+of+collagens+type+I,+II+and+III+by+Sirius+Red+and+polarization+microscopy.">Differential staining of collagens type I, II and III by Sirius Red and polarization microscopy.</a> Arch Histol Jpn. 41 (3), 267-274 (1978).</li><li>Davenport, M. L., Sherrill, T. P., Blackwell, T. S., Edmonds, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+and+inflation+of+the+mouse+lung+for+tumor+histology.">Perfusion and inflation of the mouse lung for tumor histology.</a> J Vis Exp. 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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+image+analysis+of+Picrosirius+Red+staining:+A+robust+method+for+multi-organ+fibrosis+quantification+and+characterization.">Digital image analysis of Picrosirius Red staining: A robust method for multi-organ fibrosis quantification and characterization.</a> Biomolecules. 10 (11), 1585 (2020).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>S&#233;gard, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+fibrosis+extend+and+airspace+availability+in+lung:+A+semi-automativ+ImageJ/Fiji+toolbox.">Quantification of fibrosis extend and airspace availability in lung: A semi-automativ ImageJ/Fiji toolbox.</a> PloS. 2 (2), e0298015 (2024).</li><li>Zhou, X., Moore, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+section+staining+and+microscopy.">Lung section staining and microscopy.</a> Bio Protoc. 7 (10), e2286 (2017).</li><li>Herovici, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picropolychrome:+histological+staining+technic+intended+for+the+study+of+normal+and+pathological+connective+tissue.">Picropolychrome: histological staining technic intended for the study of normal and pathological connective tissue.</a> Rev Fr Etud Clin Biol. 8, 88-89 (1963).</li><li>Sweat, F., Puchtler, H., Rosenthal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sirius+Red+F3BA+as+a+stain+for+connective+tissue.">Sirius Red F3BA as a stain for connective tissue.</a> Arch Pathol. 78, 69-72 (1964).</li><li>Cardiff, R. D., Miller, C. H., Munn, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+hematoxylin+and+eosin+staining+of+mouse+tissue+sections.">Manual hematoxylin and eosin staining of mouse tissue sections.</a> Cold Spring Harb Protoc. 2014 (6), 655-658 (2014).</li><li>Montes, G. S., Junqueira, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+the+Picrosirius-polarization+method+for+the+study+of+the+biopathology+of+collagen.">The use of the Picrosirius-polarization method for the study of the biopathology of collagen.</a> Mem Inst Oswaldo Cruz. 86, 1-11 (1991).</li><li>Ritti&#233;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+Picrosirius+Red-polarization+detection+of+collagen+fibers+in+tissue+sections.">Method for Picrosirius Red-polarization detection of collagen fibers in tissue sections.</a> Methods Mol Biol. 1627, 395-407 (2017).</li><li>Diehm, Y. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-enriched+hybrid+breast+reconstruction+reduces+risk+for+capsular+contracture+in+a+hybrid+breast+model.">Stem cell-enriched hybrid breast reconstruction reduces risk for capsular contracture in a hybrid breast model.</a> Plast Reconstr Surg. 152 (3), 572-580 (2023).</li><li>Rich, L., Whittaker, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+and+Picrosirius+Red+staining:+A+polarized+light+assessment+of+fibrillar+hue+and+spatial+distribution.">Collagen and Picrosirius Red staining: A polarized light assessment of fibrillar hue and spatial distribution.</a> Braz J Morphol Sci. 22 (2), 97-104 (2005).</li><li>Datar, U. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinicopathologic+study+of+a+series+of+giant+cell+fibroma+using+picrosirius+red+polarizing+microscopy+technique.">Clinicopathologic study of a series of giant cell fibroma using picrosirius red polarizing microscopy technique.</a> Arch Iran Med. 17 (11), 746-749 (2014).</li><li>Nishat, R., Kumar, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+fibers+in+oral+submucous+fibrosis+-+A+polarizing+microscopy+study+using+two+special+stains.">Collagen fibers in oral submucous fibrosis - A polarizing microscopy study using two special stains.</a> Indian J Pathol Microbiol. 62 (4), 537-543 (2019).</li><li>Lattouf, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picrosirius+red+staining:+a+useful+tool+to+appraise+collagen+networks+in+normal+and+pathological+tissues.">Picrosirius red staining: a useful tool to appraise collagen networks in normal and pathological tissues.</a> J Histochem Cytochem. 62 (10), 751-758 (2014).</li><li>L&#243;pez De Padilla, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picrosirius+Red+staining:+Revisiting+its+application+to+the+qualitative+and+quantitative+assessment+of+collagen+type+I+and+type+III+in+tendon.">Picrosirius Red staining: Revisiting its application to the qualitative and quantitative assessment of collagen type I and type III in tendon.</a> J Histochem Cytochem. 69 (10), 633-643 (2021).</li><li>Junqueira, L. C. U., Bignolas, G., Brentani, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picrosirius+staining+plus+polarization+microscopy,+a+specific+method+for+collagen+detection+in+tissue+sections.">Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections.</a> Histochem J. 11 (4), 447-455 (1979).</li><li>Zheng, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scar+collagen+deposition+in+the+airways+of+allografts+of+lung+transplant+recipients.">Scar collagen deposition in the airways of allografts of lung transplant recipients.</a> Am J Respir Crit Care Med. 155 (6), 2072-2077 (1997).</li><li>Astbury, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+computational+quantification+of+liver+fibrosis+is+compromised+by+inherent+staining+variation.">Reliable computational quantification of liver fibrosis is compromised by inherent staining variation.</a> J Pathol Clin Res. 7 (5), 471-481 (2021).</li><li>Feingold, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffuse+myocardial+fibrosis+among+healthy+pediatric+heart+transplant+recipients:+Correlation+of+histology,+cardiovascular+magnetic+resonance,+and+clinical+phenotype.">Diffuse myocardial fibrosis among healthy pediatric heart transplant recipients: Correlation of histology, cardiovascular magnetic resonance, and clinical phenotype.</a> Pediatr Transplant. 21 (5), 12986 (2017).</li><li>vander Ploeg, E. 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The authors have nothing to disclose.

Standard histologic methods such as Hematoxylin and Eosin, and Masson&#39;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&#39;s collagen composition.
Picrosirius Red staining was first described in 1964 to identify collagen: under transmitted light microscopy, collagen ap...

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&#39;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&#39;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&#39;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid</td>
			<td>Honeywell, Charlotte, USA</td>
			<td>33209</td>
			<td></td>
		</tr>
		<tr>
			<td>Axio Observer</td>
			<td>Zeiss, Oberkochen, Germany</td>
			<td>4633000956 (serial number)</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip 1.5</td>
			<td>Roth, Karlsruhe, Germany</td>
			<td>KCY5.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde 37%</td>
			<td>Fisher Scientific, Leicestershire, UK</td>
			<td>F/1501/PB15</td>
			<td></td>
		</tr>
		<tr>
			<td>Meyer&#39;s hemalum solution</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>109249</td>
			<td></td>
		</tr>
		<tr>
			<td>Picrosirius Red Solution</td>
			<td>Morphisto, Offenbach am Main, Germany</td>
			<td>13422</td>
			<td>alternatives that can be used: ab150681, abcam, Cambridge, UK; SRS250 ScyTek Laboratories, Logan City US</td>
		</tr>
		<tr>
			<td>Polarizing filter</td>
			<td>Zeiss, Oberkochen, Germany</td>
			<td>000000-1121-813</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary microtome, HistoCore AUTOCUT</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>149AUTO00C1, 14051956472</td>
			<td></td>
		</tr>
		<tr>
			<td>ROTI Histokitt mounting medium</td>
			<td>Roth, Karlsruhe, Germany</td>
			<td>6638.1</td>
			<td></td>
		</tr>
		<tr>
			<td>ROTI Plast Paraffin</td>
			<td>Roth, Karlsruhe, Germany</td>
			<td>6642.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotilabo-embedding cassettes, POM</td>
			<td>Roth, Karlsruhe, Germany</td>
			<td>K113.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Adhesion Microscope slide</td>
			<td>epredia, Portsmouth, UK</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Processor&#160;</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>TP 1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji software version 2.14.0/1.54f</td>
			<td>Open Source</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging Software ZEN 3.4.91</td>
			<td>Zeiss, Oberkochen, Germany</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

picrosirius red staining for semiquantitative histopathologic evaluation of collagen deposition in murine models of chronic lung allograft rejection

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.

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...

Author Spotlight: Analyzing Fibrosis Development in Chronic Lung Allograft Rejection Using Picrosirius Red Staining in Mouse Models

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.

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

Fibrosis is the pathophysiologic hallmark of chronic rejection after lung transplantation and the foremost hurdle to long-term recipient survival. Several ...

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122 Views.  University of Freiburg. 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. 

Video: Author Spotlight: Analyzing Fibrosis Development in Chronic Lung Allograft Rejection Using Picrosirius Red Staining in Mouse Models

Principles of Rodent Surgery for the New Surgeon

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform surgery on a rodent. Students, post-doctoral scholars, and others interested in performing surgery on rodents as part of a research protocol may not have had formal surgical training as part of their required coursework. Surgery itself is a technical skill, and one that will improve with practice. The principles of aseptic technique, however, often remain unexplained or untaught. For most new surgeons, this vital information is presented in piecemeal fashion or learned on the job, neither of which is ideal. It may also make learning how to perform a particular surgery difficult, as the new surgeon is learning both a surgical technique and the principles of asepsis at the same time. This article summarizes and makes recommendations for basic surgical skills and techniques necessary for successful rodent surgery. This article is designed to supplement hands-on training by the user's institution.

Before attempting surgery, a new surgeon should have training in basic surgical techniques and concepts. This article will present basic surgical considerations with an emphasis on rodents.

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-&beta;-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.

Here, we describe how to identify the stage of the murine reproductive (proestrus, estrus, metestrus, or diestrus) by simple, non-invasive collection and cytological assessment of vaginal smear samples. We further describe how vaginal cytology reflects circulating hormonal levels underlying transition through the murine reproductive cycle.

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the ...

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum &#8220;cast&#8221; intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.

Basic techniques and refinements of freeze-fracture processing of biological specimens and nanomaterials for examination by transmission electron microscopy are described. This technique is a preferred method for revealing ultrastructural features and specializations of biological membranes and for obtaining ultrastructural level dimensional and spatial data in materials sciences and nanotechnology products.

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and disease modeling, drug discovery, and regenerative medicine. To fully exploit these models, it is crucial to study them at cellular and subcellular levels. However, characterizing such in vitro 3D cell culture models can be technically challenging and requires specific expertise to perform effective analyses. Here, this paper provides detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed in vitro 3D cell culture models ranging from 100 &#181;m to several millimeters. These protocols are applicable to a wide variety of organoids and spheroids that differ in their cell-of-origin, morphology, and culture conditions. From 3D structure harvesting to image analysis, these protocols can be completed within 4-5 days. Briefly, 3D structures are collected, fixed, and can then be processed either through paraffin-embedding and histological/immunohistochemical staining, or directly immunolabeled and prepared for optical clearing and 3D reconstruction (200 &#181;m depth) by confocal microscopy.

Here, we provide detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed three-dimensional cell culture models ranging from 100 &#181;m to several millimeters, thus enabling the visualization of their morphology, cell-type composition, and interactions.

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and ...

Live Cell Imaging of Lipid Droplet Size and Fusion Using Oil Red O and BODIPY

Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells

Lipid droplets (LDs) are organelles that play an important role in lipid metabolism and neutral lipid storage in cells. They are associated with a variety of metabolic diseases, such as obesity, fatty liver disease, and diabetes. In hepatic cells, the sizes and numbers of LDs are signs of fatty liver disease. Moreover, the oxidative stress reaction, cell autophagy, and apoptosis are often accompanied by changes in the sizes and numbers of LDs. As a result, the dimensions and quantity of LDs are the basis of the current research regarding the mechanism of LD biogenesis. Here, in fatty acid-induced bovine hepatic cells, we describe how to use oil red O to stain LDs and to investigate the sizes and numbers of LDs. The size distribution of LDs is statistically analyzed. The process of small LDs fusing into large LDs is also observed by a live cell imaging system. The current work provides a way to directly observe the size change trend of LDs under different physiological conditions.

Author Spotlight: Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells  

The present protocol describes how to use oil red O to dye lipid droplets (LDs), calculate the size and number of LDs in a fatty acid-induced fatty hepatocyte model, and use BODIPY 493/503 to observe the process of small LDs fusing into large LDs by live cell imaging.

Lipid droplets (LDs) are organelles that play an important role in lipid metabolism and neutral lipid storage in cells. They are associated with a variety ...

Quantification of Skeletal Muscle Fatty Infiltration via Decellularization

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

Author Spotlight: Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic ...

Exploring HR-Dependent Cardiac Performance Using an Atrial-Pacing Protocol

A Pacing-Controlled Procedure for the Assessment of Heart Rate-Dependent Diastolic Functions in Murine Heart Failure Models

Heart failure with preserved ejection fraction (HFpEF) is a condition characterized by diastolic dysfunction and exercise intolerance. While exercise-stressed hemodynamic tests or MRI can be used to detect diastolic dysfunction and diagnose HFpEF in humans, such modalities are limited in basic research using mouse models. A treadmill exercise test is commonly used for this purpose in mice, but its results can be influenced by body weight, skeletal muscle strength, and mental state. Here, we describe an atrial-pacing protocol to detect heart rate (HR)-dependent changes in diastolic performance and validate its usefulness in a mouse model of HFpEF. The method involves anesthetizing, intubating, and performing pressure-volume (PV) loop analysis concomitant with atrial pacing. In this work, a conductance catheter was inserted via a left ventricular apical approach, and an atrial pacing catheter was placed in the esophagus. Baseline PV loops were collected before the HR was slowed with ivabradine. PV loops were collected and analyzed at HR increments ranging from 400 bpm to 700 bpm via atrial pacing. Using this protocol, we clearly demonstrated HR-dependent diastolic impairment in a metabolically induced HFpEF model. Both the relaxation time constant (Tau) and the end-diastolic pressure-volume relationship (EDPVR) worsened as the HR increased compared to control mice. In conclusion, this atrial pacing-controlled protocol is useful for detecting HR-dependent cardiac dysfunctions. It provides a new way to study the underlying mechanisms of diastolic dysfunction in HFpEF mouse models and may help develop new treatments for this condition.

Author Spotlight: Investigating HR-Dependent Cardiac Function in Mouse Models Through a Novel Atrial-Pacing Approach 

The present protocol describes obtaining the pressure-volume relationship through transesophageal pacing, which serves as a valuable tool in evaluating diastolic function&#160;in mouse models of heart failure.