Quantification of interstitial collagen content of the aortic sinus was performed as described11 (link). Briefly, serial cross-sections of 5 μm were stained with Weigert’s hematoxylin and washed in running tap water for 10 minutes followed by incubation for 4 hours in a freshly prepared 0.1% solution of Sirius Red F3B (Sigma-Aldrich, #365548) in saturated aqueous picric acid (Sigma-Aldrich, #P-6744). Sections were rinsed twice in acidified (0.01 N HCl) distilled water, dehydrated, and mounted in Permount (Fisher Scientific, #SP15–500). Polarization microscopy was used to analyze picrosirius red staining. Total collagen, thick mature orange-red fibers, and thin immature green fibers were quantified as described19 (link) using NIH ImageJ software with a defined threshold (minimum 100 and maximum 200). A mean for each mouse was calculated using the mean value of 4 sections (each 80 μm apart, beginning at the aortic valve leaflets and spanning 320 μm).
Sirius red f3b
Manufactured by Merck Group
Sourced in United States
Sirius Red F3B is a synthetic dye used in the laboratory for the staining of collagen fibers. It binds specifically to collagen and produces a bright red color, allowing for the visualization and analysis of collagen content in various tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Picric acid, by Merck Group (6 mentions)
Saturated aqueous picric acid, by Merck Group (3 mentions)
Saturated picric acid, by Merck Group (3 mentions)
Permount, by Thermo Fisher Scientific (2 mentions)
Anti mac3, by BD (2 mentions)
Picric acid solution, by Merck Group (2 mentions)
Zen 2012 v 1, by Zeiss (2 mentions)
Axio observer z1 microscope, by Zeiss (2 mentions)
Bx53 p, by Olympus (1 mentions)
Weigert s iron hematoxylin solution, by Merck Group (1 mentions)
Dpx mountant, by Merck Group (1 mentions)
Xc10 camera, by Olympus (1 mentions)
Bx60 upright microscope, by Olympus (1 mentions)
Permount mounting medium, by Thermo Fisher Scientific (1 mentions)
Rabbit antigoat horseradish peroxidase, by Agilent Technologies (1 mentions)
Goat anti type 3 collagen antibody, by Southern Biotech (1 mentions)
Bx41 microscope, by Olympus (1 mentions)
Eclipse ci light microscope, by Nikon (1 mentions)
Anti caspase 3 ab, by Cell Signaling Technology (1 mentions)
A6283, by Merck Group (1 mentions)
32 protocols using sirius red f3b
1
Quantification of Aortic Sinus Collagen
2
Quantifying Disc Degeneration via H&E and Picrosirius Red
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H&E staining was performed according to the standardized protocol. The histological grading scale system (Supplementary Table 1 ) included five categories with scores ranging from 0 points (normal) to 15 points (serious degeneration disc) using the method according to previous studies [37 (link), 38 (link)]. The picrosirius red staining method of Novais et al. [39 (link)] and Melrose et al. [40 (link)] was used after the initial removal of tissue proteoglycans by pre-digestion with bovine testicular hyaluronidase (1000 U/ml) for 2 h at 37℃. The slides were initially stained in Wiegert’s iron hematoxylin for 30 min and stained in 0.1% Sirius red F3B (26–10-8, Sigma-Aldrich) in saturated aqueous picric acid for 2 h. The slides were then dehydrated in three changes of 100% ethanol, then cleared in xylene, and mounted in an Eukitt mounting medium. The sides were examined under polarized light through the microscope (BX53P, Olympus). The percentage of thin collagen fibers (green), intermediate collagen fibers (yellow), or thick collagen fibers (red) in AF was quantified by ImageJ software in the images (average pixels from three fields per section, three sections per rat, three rats per group). Color threshold levels were maintained constant between all analyzed images. Two observers evaluated the histological score of the intervertebral disc blindly.
3
Quantitative Histological Aortic Collagen Analysis
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Formalin fixed and paraffin embedded 7 μm section of aorta with intact periaortic fat were deparaffinized and rehydrated. Aortic sections were stained with Weigert’s iron hematoxylin solution (Sigma-Aldrich) for 7 min, washed and incubated with 0.1 % Sirius red F3B (Sigma-Aldrich) for one hour in the dark. Slides were then washed in acidified water, dehydrated and mounted in DPX Mountant (Sigma-Aldrich). Quantification of adventitial collagen staining was performed using ImageJ software by blinded observers.
4
Picro-Sirius Red Staining of Frozen Tissue
5
Quantitative Histological Analysis of Ovarian Tissue
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Tissue sections were deparaffinized in histolene, rehydrated in a series of graded ethanols, then immersed in Picro-Sirius red staining solution (0.1% w/v) comprising Sirius Red F3B (Sigma-Aldrich) in a saturated aqueous solution of picric acid (Sigma-Aldrich) for 1 h at room temperature. Slides were washed four times in 0.5% glacial acetic acid for 7 min per wash. Tissues were rapidly dehydrated in 100% ethanol, cleared in histolene and mounted with DPX. The area of positive stained ovarian tissue was quantified as previously reported30 (link). Briefly, whole tissue section images were captured on the DotSlide system at ×20 objective using an XC10 camera (Olympus). ImageJ was used to quantify the area of positive staining above a threshold that was set based on the staining in the oldest animal. This threshold was kept constant for all images analysed. Four tissue sections per ovary per animal at d7 + 12 h were analysed (n = 4–5/age/treatment).
6
Sirius Red Staining for Fibrosis
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To evaluate fibrosis, 10 µm thick frozen sections were air dried for 10 to 15 minutes, fixed in cold acetone (−20°C) for 10 minutes, and stained for collagen in 0.1% v/v solution of Sirius red F3B (Sigma-Aldrich) saturated with picric acid (Sigma-Aldrich) for 60 minutes. The sections were then washed for 2 minutes in 0.5% v/v glacial acetic acid, and then treated as described above.
7
Cardiac Fibrosis and Hypertrophy Analysis
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The anesthetized animals were sacrificed by exsanguination, heart and lung were washed with PBS at 4°C, harvested and processed for analysis. The RV was separated from the left ventricle (LV) and septum (S), and all parts were weighed separately. The degree of RV hypertrophy was calculated as the RV/LV+S ratio.
The degree of cardiac fibrosis was determined by Sirius red staining. Briefly, 10 µm-thick paraffin-embedded tissue sections were deparaffinized, rehydrated and stained in iron hematoxylin for 10 min, followed by rinsing in water for 10 min. The slides were then stained in 0.1% (w/v) Sirius Red F3B (Sigma-Aldrich, Buchs SG, Switzerland) in saturated aqueous picric acid (Sigma-Aldrich, Buchs SG, Switzerland) for 1 h. Sections were washed twice in 0.5% v/v acetic acid, dehydrated rapidly 3 times in 100% alcohol, cleared in xylene and mounted using Merckoglas medium (Merck, Germany). Each section was photographed under a light microscope (Nikon Instruments Inc., Melville, NY, USA) at 10x magnification. At least 10 fields were randomly selected for each rat, each of which contains at least 6 vessels. Images were analyzed using Image software. The percentage collagen was calculated as fibrosis area/total area of the tissue section.
The degree of cardiac fibrosis was determined by Sirius red staining. Briefly, 10 µm-thick paraffin-embedded tissue sections were deparaffinized, rehydrated and stained in iron hematoxylin for 10 min, followed by rinsing in water for 10 min. The slides were then stained in 0.1% (w/v) Sirius Red F3B (Sigma-Aldrich, Buchs SG, Switzerland) in saturated aqueous picric acid (Sigma-Aldrich, Buchs SG, Switzerland) for 1 h. Sections were washed twice in 0.5% v/v acetic acid, dehydrated rapidly 3 times in 100% alcohol, cleared in xylene and mounted using Merckoglas medium (Merck, Germany). Each section was photographed under a light microscope (Nikon Instruments Inc., Melville, NY, USA) at 10x magnification. At least 10 fields were randomly selected for each rat, each of which contains at least 6 vessels. Images were analyzed using Image software. The percentage collagen was calculated as fibrosis area/total area of the tissue section.
8
Sirius Red Staining for Liver Fibrosis
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Sirius Red staining was performed on formalin-fixed paraffin-embedded liver sections (2 μm thick) with rapid exposure to Harry’s hematoxylin to stain nuclei after staining in 0.1% Sirius Red F3B (Sigma—Aldrich, St. Louis, MO, USA). Computer based morphometric quantification of liver fibrosis in groups G1-G5 was then performed [23 (link)].
9
Sirius Red Staining for Collagen
10
Collagen Staining in Breast Tumors
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Sectioned breast tumor slides were hydrated and incubated for 1 h with 0.1% Sirius Red F3B (Sigma-Aldrich, Oakville, ON, Canada) dissolved in a saturated aqueous solution of picric acid, washed twice with 0.5% acetic acid and then dehydrated [55 (link)]. Collagen type I, III and IV were stained red against a pale orange background when visualized under light microscope. Areas of positive staining were quantified using Adobe Photoshop (Adobe Inc., San Jose, CA, USA) and Image J software. The results were averaged from 10 image fields for each sample.
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