Osteogenic differentiation was assessed using alcian blue staining. Briefly, tissue was embedded in paraffin and sections (4.0 µm thick) were mounted onto glass slides, cleared using xylene, and gradually rehydrated. Antigen retrieval was performed on slides using sodium citrate buffer (10 mM sodium citrate and pH 6) at 100°C for 20 min. Slides were cooled and rinsed in distilled water to remove citrate solution. Slides were blocked using Super Block (Thermo Fisher Scientific, Inc.) for 1 h at room temperature. For slides stained with alcian blue staining, sections were stained with alcian blue solution (Sigma-Aldrich, Merck KGaA) and Weigert's Iron Hematoxylin (Sigma-Aldrich, Merck KGaA) according to the manufacturer's protocol. Alcian blue Stain kit (Connective Tissue Stain; Abcam) was also used to stain sections according to the manufacturer. For slides stained with hematoxylin and eosin (H&E), sections were cleared and rehydrated followed by staining with H&E, according to standard protocols. Sections were then dehydrated gradually and mounted for imaging. All images were acquired using a Nikon Digital Imaging System.
Weigert s iron hematoxylin
Manufactured by Merck Group
Sourced in United States
Weigert's iron hematoxylin is a staining solution used in histology and microscopy. It is a mixture of hematoxylin and iron salts, which provides a blue-black staining of nuclei and other cellular structures.
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Lab products found in correlation
Safranin o, by Merck Group (5 mentions)
Fast green, by Merck Group (5 mentions)
Safranin o solution, by Merck Group (4 mentions)
Aniline blue, by Merck Group (2 mentions)
Phosphomolybdic phosphotungstic acid, by Merck Group (2 mentions)
Entellan, by Merck Group (2 mentions)
Fast green fcf, by Merck Group (2 mentions)
Xylene, by Merck Group (2 mentions)
Fast green solution, by Merck Group (2 mentions)
Superblock, by Thermo Fisher Scientific (1 mentions)
Digital imaging system, by Nikon (1 mentions)
Alcian blue solution, by Merck Group (1 mentions)
Methyl methacrylate, by Merck Group (1 mentions)
Masson s trichrome staining, by Merck Group (1 mentions)
Polycut e microtome, by Leica (1 mentions)
Chromotrope 2r, by Merck Group (1 mentions)
Oil red o, by Merck Group (1 mentions)
Biebrich scarlet acid fusion, by Merck Group (1 mentions)
Nh4oh, by Merck Group (1 mentions)
Harris hematoxylin, by Merck Group (1 mentions)
25 protocols using weigert s iron hematoxylin
1
Histological assessment of osteogenic differentiation
2
Histological analysis of bone samples
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Bones were fixed overnight at 4°C in 70% ethanol solution and dehydrated in a graded ethanol series. Undecalcified samples were embedded in methyl methacrylate (Merck, Rahway, NJ). Serial sections, 4 μm thick, were cut on a microtome (Polycut E microtome, Leica, Wetzlar, Germany). Series of consecutive sections representative of micro-CT images were stained respectively with toluidine blue (pH 3.8), von Kossa reagent (5% silver nitrate solution, Sigma-Aldrich, St Louis, MO), Masson’s trichrome staining (Sigma-Aldrich, St Louis, MO) and safranin O staining (Weigert’s iron hematoxylin, Sigma-Aldrich, St Louis, MO; Fast Green, Prolabo, Paris, France; and Safranin O solution, Sigma-Aldrich, St Louis, MO).
3
Trichrome Staining Protocol
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Tissue sections were deparaffinized in xylene and hydrated to a decrescent series of ethanol until distilled water. Then the slides were treated with Weigert’s iron hematoxylin (Sigma Aldrich, Saint Louis, MO, USA) stain from 7 min. Slides were placed into Chromotrope 2R (Sigma Aldrich, Saint Louis, MO, USA) at 0.2% with Light green at 0.8% solution for 5 min. Then, a quickly rinse off the stain with distilled water was performed and then differentiated with acetic acid at 0.5%. Tissue sections were then dehydrated, cleared, and mounted. The cytoplasm of the cells was stained in red, fibrin in pink whereas collagen was observed in green, in contrast with blue or black nuclei. All samples were examined by light microscopy using a Zeiss microscope Mod. Axioplan 2 (Göttingen, Germany)
4
Evaluating 17α-E2 Effects on Liver Lipids and Fibrosis
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To evaluate the effects of 17α-E2 treatment on lipid accumulation and fibrosis, we evaluated fixed liver tissue. Tissues were fixed in 4% PFA for 24 hr, cryo-embedding samples were transferred to 30% sucrose for 72 hr and embedded in OCT, paraffin-embedding samples were transferred to 1X PBS for 48 hr, then to 70% ethanol until embedding. Liver Oil-red-O and Masson’s trichrome staining were performed by the Oklahoma Medical Research Foundation Imaging Core Facility using previously reported methodology (Leonard et al., 2018 (link); Mehlem et al., 2013 (link)). Oil-red-O (ORO, Sigma-Aldrich, St. Louis, MO) and H and E counterstaining were performed on cryo-embedded tissues, and were imaged within 6 hr of staining. Red lipid stain was blindly quantified from 10 images per animal using ImageJ software and presented as a lipid to total tissue ratio. Masson’s trichrome staining was performed on paraffin embedded liver tissue and was used for qualitative purposes. In brief, slides were stained with Weigert’s Iron Hematoxylin (Sigma-Aldrich, St. Louis, MO), washed, stained with Biebrich Scarlet-Acid Fusion (Sigma-Aldrich, St. Louis, MO), washed, stained with Phosphomolybdic Acid-Phosphotunsctic Acid, and then stained with Aniline Blue (Sigma-Aldrich, St. Louis, MO).
5
Joint Tissue Histological Staining and Scoring
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Joint tissue sections were deparaffinized in xylene for 15 min and hydrated in an ethanol series. For H&E staining, the sections were incubated with Harris hematoxylin (Sigma-Aldrich) for 10 min, washed in tap water, and dipped sequentially in 1% HCl, 0.2% NH4OH, and eosin (Sigma-Aldrich) for 90 sec and washed. For safranin O staining, the sections were incubated in Weigert's iron hematoxylin (Sigma-Aldrich) for 10 min, washed in tap water for 10 min, incubated in Fast green (Sigma-Aldrich) for 5 min, dipped in 1% acetic acid 2 to 3 times, incubated in safranin O (Sigma-Aldrich) for 5 min and washed. For toluidine blue staining, the sections were incubated in toluidine blue for 3 min. Each stained section was subsequently washed in tap water, dehydrated in an ethanol series, dipped in xylene, and mounted. Inflammation and joint destruction scores were measured by three independent investigators. Inflammation scores were measured by adding scores, graded as 0-3, were based on the layer status of synovial membranes and scores, graded as 0-3, were based on the infiltration of lymphocytes. Joint destruction scores were measured by adding scores, graded as 0-3, were based on cartilage erosion, and scores, graded as 0-3, were based on pannus invasion into the cartilage. The detailed scoring method was described in reference study (19 (link)).
6
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.
7
Cardiac Morphometric Analysis via Histology
8
Histological Analysis of FFPE Tissue
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5-µm thick formalin-fixed, paraffin-embedded (FFPE) sections were briefly de-paraffinized at room temperature in four washes of xylene (3 min each). Sections were hydrated in graded ethanols (100%, 100%, 95%, 95%, 70%; all 1 min) to distilled water. Nuclei were stained using immersion in Weigert's iron hematoxylin (Sigma-Aldrich, St. Louis, MO) for 8 mins, followed by washing in running tap water for 10 mins. Sections were stained at room temperature in a solution of 0.1% Sirius red F3BA in saturated aqueous picric acid (Sigma-Aldrich) for one hour, as described in other studies [13] , [14] (link). Subsequently, sections were rinsed in two washes of a 0.5% acetic acid in distilled water solution (5 min each). Ascending concentrations of ethanol were used for dehydration (70%, 95%, 95%, 100%, 100%; 4 quick dips each) and sections were cleared in three washes of xylene (4 quick dips). Sections were covered with resinous mounting medium and glass cover slips.
9
Evaluating Cartilage Degeneration via Histology
10
Histological Evaluation of Rat Joints
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After micro-CT analysis, the hind paws and tails of the rats were decalcified with OsteoSoft decalcifier solution (no. 101728; Merck, Burlington, MA, USA) and embedded in paraffin. Five micrometer sections were stained for hematoxylin and eosin (H&E) or Safranin O Fast Green. For H&E staining, sections were incubated in Mayer’s hematoxylin (no. MHS16) and Eosin Y solution (no. HT110116) (both from Sigma, St. louis, MO, USA). For Safranin O Fast Green staining, sections were incubated in Weigert’s iron hematoxylin (no. HT1079), 0.1% fast green FCF (no. F7252), and 0.1% Safranin O solution (no. HT90432) (all from Sigma, St. louis, MO, USA). The sections were subsequently dehydrated and embedded in Entellan (no. 1.07961.0100; Merck, Burlington, MA, USA). Separate experimentally blinded observers scored the caudal spine, calcaneus, and tarsal regions semi-quantitatively (0–4) for inflammation, bone proliferation and pannus formation.
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