For histological analysis, WT and galectin-3−/− mice were sacrificed 60 days after pristane injection. Several oil granulomas were excised, washed in cold saline, fixed in buffered 10 % formalin for 24 h at 4 °C, embedded in paraffin and sectioned into 4-m thick slices. The slices were dewaxed using xylene, hydrated in a graded ethanol, and then washed with distilled water. Lastly, the slices were processed routinely with hematoxylin & eosin (H&E) or with Masson’s Trichrome stain. For Masson’s Trichrome stain, briefly, slides were placed in Bouin solution for 1 h at 56 °C, then stained with Weigert hematoxylin (10 min; Sigma-Aldrich, St. Louis, MO), followed by Biebrich scarlet acid fuchsin (5 min; Sigma-Aldrich), phosphomolybdic/phosphotungstic acid solution (10 min; Sigma-Aldrich), and aniline blue (5 min; Sigma-Aldrich).
Biebrich scarlet acid fuchsin
Manufactured by Merck Group
Sourced in United States
Biebrich scarlet acid fuchsin is a laboratory reagent used as a staining dye. It is a mixture of two anionic dyes, Biebrich scarlet and acid fuchsin, which are commonly used in histological and cytological staining techniques.
Automatically generated - may contain errors
Lab products found in correlation
Aniline blue, by Merck Group (6 mentions)
Phosphomolybdic phosphotungstic acid solution, by Merck Group (5 mentions)
Bouin s solution, by Merck Group (3 mentions)
Weigert hematoxylin, by Merck Group (2 mentions)
Phosphomolybdic phosphotungstic acid, by Merck Group (2 mentions)
Hematoxylin, by Merck Group (2 mentions)
Aniline blue solution, by Merck Group (2 mentions)
Weigert s hematoxylin, by Merck Group (1 mentions)
Neutral buffered formalin solution, by Merck Group (1 mentions)
Mounting medium, by Merck Group (1 mentions)
Weigert s iron hematoxylin, by Merck Group (1 mentions)
Direct red 80 in saturated picric acid, by Merck Group (1 mentions)
Oil red o stain, by Merck Group (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Accustain trichrome stains, by Merck Group (1 mentions)
Poly mount, by Polysciences (1 mentions)
Biotinylated anti mouse igg, by Vector Laboratories (1 mentions)
H e staining kit, by Abcam (1 mentions)
Fast green, by Merck Group (1 mentions)
Weigert iron hematoxylin, by Merck Group (1 mentions)
12 protocols using biebrich scarlet acid fuchsin
1
Histological Analysis of Pristane-Induced Granulomas
2
Masson Staining for Liver Fibrosis
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Collagen deposition in the liver was evaluated by Masson staining. All procedures were performed at room temperature unless otherwise specified. Liver tissues were fixed with 10% neutral formalin for 48-72 h, embedded in paraffin, continuously sectioned at a thickness of 5 µm, and stained with Masson stains. Briefly, slides were placed in Bouin solution (Richard Allen Scientific; Thermo Fisher Scientific, Inc.) for 1 h at 56°C, after which, the slides were stained with Weigert hematoxylin (Sigma-Aldrich; Merck KGaA) for 10 min, followed by Biebrich scarlet-acid fuchsin for 5 min, phosphomolybdic/phosphotungstic acid solution for 10 min and aniline blue (all Sigma-Aldrich; Merck KGaA) for 5 min. Hepatic fibrosis was graded based on the internationally used Metavir scoring system (31 (link)).
3
Histological Analysis of Liver in Infectious Disease
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For histological analyses, animals were sacrificed after 90 days of infection. Livers were removed, cut into 0.5 mm thick slices, washed in cold saline and fixed in Bouin’s fixative. After 6 h of fixation, specimens were dehydrated in alcohol, and embedded in paraffin. Sections of 5 μm were obtained and stained with Hematoxylin-eosin and Masson’s trichrome staining.
For Masson’s Trichrome stain, briefly, slides were placed in Bouin’s solution for 1 h at 56 °C, then stained with Weigert’s hematoxylin (10 min; Sigma-Aldrich, St. Louis, MO), followed by Biebrich scarlet acid fuchsin (5 min; Sigma-Aldrich), phosphomolybdic/phosphotungstic acid solution (10 min; Sigma-Aldrich), and aniline blue (5 min; Sigma-Aldrich).
For α-smooth muscle actin (α-SMA) immunofluorescence, paraffin-embedded sections were dewaxed and hydrated and washed several times in PBS and further treated with 50nM NH4Cl (30 min), 0.05 % saponin/0.1 M PBS (30 min) and 0.05 % gelatin/0.05 % saponin/PBS (30 min). Cells were incubated with human monoclonal anti-SMA (Sigma-Aldrich, Inc., St. Louis, MO) diluted 1:400 in 0.05 % gelatin/0,05%saponin/PBS as the primary antibody at 4 °C, overnight. Thereafter, sections were incubated with anti-human Alexa Fluor® 546 (Life Technologies, Inc.) diluted 1:1000. Sections were examined by confocal microscopy Olympus FV-1000.
For Masson’s Trichrome stain, briefly, slides were placed in Bouin’s solution for 1 h at 56 °C, then stained with Weigert’s hematoxylin (10 min; Sigma-Aldrich, St. Louis, MO), followed by Biebrich scarlet acid fuchsin (5 min; Sigma-Aldrich), phosphomolybdic/phosphotungstic acid solution (10 min; Sigma-Aldrich), and aniline blue (5 min; Sigma-Aldrich).
For α-smooth muscle actin (α-SMA) immunofluorescence, paraffin-embedded sections were dewaxed and hydrated and washed several times in PBS and further treated with 50nM NH4Cl (30 min), 0.05 % saponin/0.1 M PBS (30 min) and 0.05 % gelatin/0.05 % saponin/PBS (30 min). Cells were incubated with human monoclonal anti-SMA (Sigma-Aldrich, Inc., St. Louis, MO) diluted 1:400 in 0.05 % gelatin/0,05%saponin/PBS as the primary antibody at 4 °C, overnight. Thereafter, sections were incubated with anti-human Alexa Fluor® 546 (Life Technologies, Inc.) diluted 1:1000. Sections were examined by confocal microscopy Olympus FV-1000.
4
Histological Examination of Aortic Roots
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The extracted aortic roots from the mice specimens were processed for histological examination. The specimens were first fixed in 10% neutral buffered formalin solution (Sigma-Aldrich) and subsequently embedded in optimal cutting temperature (OCT) compound (Sakura Finetek). The lipid accumulation within the aortic root sections, cut at 10 µm thickness with a cryostat, was visualized using Oil Red O stain (Sigma-Aldrich). Hematoxylin (Sigma-Aldrich) served as a counterstain to highlight the nuclei.
To assess collagen content, the aortic root sections underwent Masson’s Trichrome and Sirius Red staining protocols, respectively. In Masson’s Trichrome staining, the sections were sequentially treated with Weigert’s iron Hematoxylin (Sigma-Aldrich), Biebrich scarlet-acid fuchsin (Sigma-Aldrich), phosphomolybdic-phosphotungstic acid (Sigma-Aldrich), and aniline blue (Sigma-Aldrich). Sirius Red staining involved an incubation period of one hour in Sirius Red solution (Direct Red 80 in saturated picric acid, Sigma-Aldrich) followed by a wash in acidified water. Post-staining, the sections were dehydrated, cleared, and affixed with a mounting medium (Sigma-Aldrich). The stained sections were subsequently observed under a light microscope and images were captured for further quantitative evaluation.
To assess collagen content, the aortic root sections underwent Masson’s Trichrome and Sirius Red staining protocols, respectively. In Masson’s Trichrome staining, the sections were sequentially treated with Weigert’s iron Hematoxylin (Sigma-Aldrich), Biebrich scarlet-acid fuchsin (Sigma-Aldrich), phosphomolybdic-phosphotungstic acid (Sigma-Aldrich), and aniline blue (Sigma-Aldrich). Sirius Red staining involved an incubation period of one hour in Sirius Red solution (Direct Red 80 in saturated picric acid, Sigma-Aldrich) followed by a wash in acidified water. Post-staining, the sections were dehydrated, cleared, and affixed with a mounting medium (Sigma-Aldrich). The stained sections were subsequently observed under a light microscope and images were captured for further quantitative evaluation.
5
Histological Analysis of Bladder Composition
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To evaluate the proportion of collagen and smooth muscle, the bladder walls of
rats in each group were stained with Masson trichrome. Bladder wall sections
were deparaffinized, rehydrated with graded alcohols, immersed in warm Bouin’s
solution (Sigma, St. Louis, MO, USA) (55 to 60°C) for 2 h and washed under
running tap water for 2 min followed by distilled water for 30 to 60 s. They
were stained with Weigert Hematoxylin (Merck, Darmstadt, Germany) for 10 min and
washed out. Subsequently, the smooth muscle was stained red with Biebrich
Scarlet-Acid-Fuchsin (Sigma, St. Louis, MO, USA) for 10 min, rinsed and immersed
in phosphomolybdic phosphotungstic acid (Sigma, St. Louis, MO, USA) for 15 min.
The collagen was stained blue with Aniline Blue (Sigma, St. Louis, MO, USA) for
10 min and rinsed in distilled water followed by immersion in 1% acetic acid for
5 min. Finally, the tissues were rehydrated with 100% ethanol, left to air dry
and mounted. All sections of bladder tissue were stained at the same time.
rats in each group were stained with Masson trichrome. Bladder wall sections
were deparaffinized, rehydrated with graded alcohols, immersed in warm Bouin’s
solution (Sigma, St. Louis, MO, USA) (55 to 60°C) for 2 h and washed under
running tap water for 2 min followed by distilled water for 30 to 60 s. They
were stained with Weigert Hematoxylin (Merck, Darmstadt, Germany) for 10 min and
washed out. Subsequently, the smooth muscle was stained red with Biebrich
Scarlet-Acid-Fuchsin (Sigma, St. Louis, MO, USA) for 10 min, rinsed and immersed
in phosphomolybdic phosphotungstic acid (Sigma, St. Louis, MO, USA) for 15 min.
The collagen was stained blue with Aniline Blue (Sigma, St. Louis, MO, USA) for
10 min and rinsed in distilled water followed by immersion in 1% acetic acid for
5 min. Finally, the tissues were rehydrated with 100% ethanol, left to air dry
and mounted. All sections of bladder tissue were stained at the same time.
6
Immunohistochemical Staining of Tropomyosin and Collagen in BAMs
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BAMs were fixed in 4% formaldehyde for 1 h and rinsed 3 × 5 min in PBS.
For tropomyosin staining, samples were permeabilized with methanol, incubated with mouse anti-tropomyosin antibody (Sigma #T9283) 1:100, followed by a secondary biotinylated anti-mouse IgG and then a preformed avidin and biotinylated horse radish peroxidase complex (Vector Laboratories, Burlingame, CA, United States). Development was by addition of DAB to produce a brown precipitate. Fixed BAMs were sent out for cross-sectional sectioning and hematoxylin and eosin staining (Mass Histology Service, Worcester, MA, United States). Slides were deparaffinized and then gradually rehydrated from 100% ethanol to H2O and stained for collagen using Accustain Trichrome Stains (Masson) (Sigma-Aldrich). The Trichome protocol was adapted as follows. Slides were soaked overnight in Bouin’s solution (Sigma), stained in hematoxylin (Sigma), and then rinsed in running tap water. The slides were then placed in Biebrich scarlet acid fuchsin (Sigma), followed by 12 min in a working phosphotungstic/phosphomolybdic acid solution (Sigma), and then 6 min in an aniline blue Solution (Sigma). Sections were rinsed in double distilled water in between every staining step. The slides were then destained for 1 min using a 1% acetic acid solution (Sigma), dehydrated with ethanol, and mounted in Polymount (Polysciences Inc.).
For tropomyosin staining, samples were permeabilized with methanol, incubated with mouse anti-tropomyosin antibody (Sigma #T9283) 1:100, followed by a secondary biotinylated anti-mouse IgG and then a preformed avidin and biotinylated horse radish peroxidase complex (Vector Laboratories, Burlingame, CA, United States). Development was by addition of DAB to produce a brown precipitate. Fixed BAMs were sent out for cross-sectional sectioning and hematoxylin and eosin staining (Mass Histology Service, Worcester, MA, United States). Slides were deparaffinized and then gradually rehydrated from 100% ethanol to H2O and stained for collagen using Accustain Trichrome Stains (Masson) (Sigma-Aldrich). The Trichome protocol was adapted as follows. Slides were soaked overnight in Bouin’s solution (Sigma), stained in hematoxylin (Sigma), and then rinsed in running tap water. The slides were then placed in Biebrich scarlet acid fuchsin (Sigma), followed by 12 min in a working phosphotungstic/phosphomolybdic acid solution (Sigma), and then 6 min in an aniline blue Solution (Sigma). Sections were rinsed in double distilled water in between every staining step. The slides were then destained for 1 min using a 1% acetic acid solution (Sigma), dehydrated with ethanol, and mounted in Polymount (Polysciences Inc.).
7
Histological Analysis of Rat Knee Joints
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After euthanization using an overdose of sodium pentobarbital, rat knee joints and posterior capsule tissues were removed, fixed with 4% paraformaldehyde, decalcified in 10% EDTA, and then embedded in paraffin. Samples were embedded in paraffin and cut into 4-μm thick sections. Hematoxylin-eosin (HE) staining and Masson trichrome staining were used for qualitative observation. The tissues were deparaffinized and hydrated in distilled water and then covered with hematoxylin and eosin, respectively, by using an HE staining kit (Abcam). Masson staining was conducted using tissue covered by Weigert iron hematoxylin, Biebrich scarlet-acid fuchsin and aniline blue (Sigma-Aldrich) according to the manufacturer’s instructions.
8
Histological Analysis of Gill Tissues
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Fresh gill tissues collected from the experimental and control animals were immediately submerged in fixing solution (10 mL of formaldehyde, 1 g of calcium chloride, and 90 mL of double-distilled water) overnight for fixation. The tissue was then embedded in paraffin, sectioned into 4 μmol/L slices, dried, dewaxed, and washed with water. The sections were then treated with Biebrich Scarlet-Acid Fuchsin (Sigma, USA) for 17 h. After that, the slides were washed with water and placed in 70%, 80%, and 95% alcohol for 30 s sequentially for dehydration. The sections were subsequently stained with 0.1% Fast-Green (Sigma, USA) in 1×PBS for 20 s. After twice washing with water, the sections were immersed for 5 min in each of the following: 100% alcohol, 100% alcohol, alcohol: xylene (1:1). After complete air-drying, the slides were observed and photographed under a light microscope. Quantification of mitochondrial number was performed using ImageJ software.
9
Paraffin Section Histological Staining
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Paraffin sections of the left lateral lobe were de-paraffinized and hydrated with deionized water and stained with Bouin’s Solution, Wiegerts Iron Hematoxylin and Biebrich Scarlet-Acid Fuchsin as per manufacturer’s instructions (Sigma-Aldrich).
10
Masson's Trichrome Staining for Colon Fibrosis
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Masson’s trichrome staining for the detection of collagen fibers in colon tissue was done according to the method described earlier [48 (link)]. Paraffin slides were deparaffinized and rehydrated with xylene and ethanol. Following washing with water, these sections were treated with Bouin’s solution (HT10132, Sigma-Aldrich) at 56 °C for 1 h and incubated with Weigert iron hematoxylin (HT1079, Sigma-Aldrich) for 10 min. After washing with water, these sections were treated with Biebrich Scarlet-acid fuchsin (HT151, Sigma-Aldrich) and next incubated in phosphomolybdic-phosphotungstic acid solution (HT153, Sigma-Aldrich) for 10–15 min. These sections were incubated with aniline blue solution (B8563, Sigma-Aldrich) for 5 min and next with light-green solution for 1 min. After washing, these sections were incubated in glacial acetic acid solution for 5 min, then dehydrated quickly in 95% ethanol and then washed in xylene. Coverslips were covered onto the slides and mounted by Permount® (Thermo Fisher Scientific). The fibrosis analysis was conducted according to the previously described method [49 (link)].
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