After fixation of the constructs in 10% neutral buffered formalin at room temperature for 20 min and 5 times washing with UPW, samples were dehydrated and impregnated with paraffin over night by use of a tissue processor (TPC 15 Duo, Medite AG, Winter Garden FL, United States) before being embedded in paraffin using a paraffin embedding station (TES 99, Medite AG, Winter Garden, FL, USA). Paraffin sections of 6–7 µm (HM355S, Microm International, Walldorf, Germany) were stained with Haematoxylin and Eosin (H&E) (Sigma-Aldrich, Buchs SG, Switzerland) for a general overview. Collagen distribution was visualised by Sirius Red staining while mineralisation was shown with von Kossa staining (Sigma-Aldrich, Buchs SG, Switzerland).
Haematoxylin and eosin h e
Manufactured by Merck Group
Sourced in United States
Haematoxylin and eosin (H&E) is a common staining technique used in histology and pathology laboratories. Haematoxylin stains cell nuclei blue, while eosin stains various cellular and extracellular structures pink or red. This combination of stains allows for the visualization of different tissue components under a microscope, enabling the assessment of tissue morphology and structure.
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Lab products found in correlation
Von kossa staining, by Merck Group (1 mentions)
Masson s trichrome dye, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Leishman s staining solution, by Merck Group (1 mentions)
Cm h2dcfda, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Merck Group (1 mentions)
Sinapic acid, by Merck Group (1 mentions)
Dopamine hydrochloride da, by Merck Group (1 mentions)
Masson s trichrome mt staining kit, by Merck Group (1 mentions)
Nahco3, by Merck Group (1 mentions)
Na2co3, by Merck Group (1 mentions)
Quercetin, by Merck Group (1 mentions)
Isopropanol, by Merck Group (1 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (1 mentions)
Agno3, by Merck Group (1 mentions)
Zoletil 50, by Virbac (1 mentions)
Decalcifying solution lite, by Merck Group (1 mentions)
Safranin o and fast green, by Merck Group (1 mentions)
Dm750 microscope, by Leica camera (1 mentions)
Escherichia coli e coli lps, by Merck Group (1 mentions)
12 protocols using haematoxylin and eosin h e
1
Histological Analysis of Tissue Samples
2
Histopathological Evaluation of NASH
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After fixation and embedding, five hepatic tissue samples in each group were sectioned at 4 μm thickness and stained with haematoxylin and eosin (HE) (Sigma, St. Louis, MO). An experienced pathologist evaluated all histological changes, including inflammation and hepatocellular damage in a blinded manner under light microscopy. The total score ranges from 0 to 10 (Hu 2012 (link)), which is based on a semi-quantitative analysis of the four definer criteria of non-alcoholic steatohepatitis: steatosis (0–2), cholestasis (0–1), apoptotic (0–2) and lobular inflammation (0–5).
3
Histological Analysis of Murine Hearts
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Whole hearts isolated from 32-week-old mice (NAGLU-/-, n = 5 and WT, n = 5) were fixed overnight in a bath of 4% aqueous buffered formalin, and processed for paraffin embedding. Coronal sections (10 μm thick) containing both right and left ventricles were processed as previously described [17 (link)–18 (link)]. Sequential sections from each heart were stained with haematoxylin and eosin (H&E) (Sigma-Aldrich), Masson's trichrome dye (Sigma-Aldrich) and periodic acid-Schiff Alcian blue-PAS (Dako). All sections were examined on a light microscope (Leitz, DIAPLAN), and images were acquired with a digital camera (Digital JVC, TK-C1380).
Plastic embedded sections were obtained from heart tissue specimens of 32-week-old mice fixed in 2.5% glutaraldehyde and postfixed in 1% osmium tetroxide in cacodylate buffer (0.2 M, pH 7.4). Tissues were washed and dehydrated in a graded series of ethanol solutions, cleared in propylene oxide, and embedded in Epon—Araldite resin. Semithin (0.5 μm) sections were stained with toluidine blue and examined under a light microscope.
Plastic embedded sections were obtained from heart tissue specimens of 32-week-old mice fixed in 2.5% glutaraldehyde and postfixed in 1% osmium tetroxide in cacodylate buffer (0.2 M, pH 7.4). Tissues were washed and dehydrated in a graded series of ethanol solutions, cleared in propylene oxide, and embedded in Epon—Araldite resin. Semithin (0.5 μm) sections were stained with toluidine blue and examined under a light microscope.
4
Cytotoxicity and Oxidative Stress Assessment
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B[a]P of HPLC grade was acquired from Invitrogen for the present investigation. The below listed chemicals were procured from Sigma-Aldrich: sinapic acid, leishman’s staining solution, safranin staining solution, diffquick solutions, haematoxylin and eosin (H&E), Dulbecco’s modified eagle’s medium (DMEM), antimycotic mixtures, fetal bovine serum (FBS), 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT), and CM-H2DCFDA. Other chemicals were assimilated in this investigation from Himedia, USA.
5
Multimodal Characterization of Tissue Samples
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NaHCO3, Na2CO3, AgNO3, dopamine hydrochloride (DA), quercetin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), isopropanol, methoxyl-, amine-functionalised silicone, haematoxylin and eosin (H&E), and Masson’s trichrome (MT) staining kit were purchased from Sigma-Aldrich, USA. The optimal cutting temperature (OCT) compound known as the inert support medium for cryo-sectioning was obtained from ThermoFisher Scientific, USA. The Zoletil50® was supplied by Virbac, Vietnam. Ferric chloride (FeCl3), ethanol, acid acetic and formal aldehyde were purchased from Xilong Chemical Ltd, China. The mice used in the in vivo studies were supplied by the Pasteur Institute, Ho Chi Minh City, Vietnam.
6
Histological Analysis of Mouse Knee Joints
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One leg was randomly selected from each mouse and dissected for histology. The knee joints were collected and fixed in 4% paraformaldehyde solution. Fixed joints were decalcified in Decalcifying Solution Lite (Sigma‐Aldrich, St. Louis, MO, USA) for 6 h and embedded in paraffin. Joint tissues were sectioned by a microtome, and sections (5 μm) had been hydrated in 100%, 90%, 70%, and 50% ethanol in distilled water (DW) for 5 min each. Hydrated samples were stained with haematoxylin and eosin (H&E) (Sigma‐Aldrich, St. Louis, MO, USA) for morphological analysis and measurement of the infiltration of immune cells, or safranin O and fast green (Sigma‐Aldrich, St. Louis, MO, USA) for determining cartilage damage. Stained samples were dehydrated in 50%, 70%, 90% and 100% ethanol and xylene and mounted by using balsam. They were observed by using DM750 microscope (Leica, Wetzlar, Germany).
7
Osteoclastogenesis Induction Protocol
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Recombinant human colony-stimulating factor 1 (rhCSF1), TNFSF11 (rhTNFSF11) and MCP-1 (rhMCP-1) were purchased from PeproTech, Inc. α-minimal essential medium (α-MEM), penicillin-streptomycin solution, PBS without calcium and magnesium, FBS, DAPI staining solution and Rhodamine-conjugated phalloidin were purchased from Gibco; Thermo Fisher Scientific, Inc. The tartrate-resistant acid phosphatase (TRAP) staining kit, Escherichia coli (E. coli) LPS, and haematoxylin and eosin (H&E) were purchased from Sigma-Aldrich; Merck KGaA. Cell Counting Kit-8 (CCK-8) reagent was obtained from Dojindo Molecular Technologies, Inc. 7ND protein was purified as previously reported (12 (link)).
8
Histomorphometric Analysis of Intestinal Tissue
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After 42 h of fixation in 10% chilled formalin solution, the intestines were dehydrated in increasing gradients of ethanol (50%, 65%, 75%, 85%, 95%, and 100%). The samples were embedded in hydroxyethyl methacrylate (Historesin mounting medium, Leica, Germany) for three days. A series of ~5–10 histological sections were performed on each sample (section thickness ~5 µm), mounted on slides, and stained using Haematoxylin and Eosin (H&E; Sigma Aldrich, St. Louis, MO, USA). Image acquisition was performed using a Zeiss Axioplan 2 microscope mounted with a Nikon (Tokyo, Japan) digital camera DXM 1200F. The external circumference of the serosa, mucosal height, and muscularis layer thickness were quantified using ImageJ (Fiji) software, version IJ 1.46r, Wisconsin, USA, [36 (link)]. The number of goblet cells was determined by manually counting them in the complete mucosal region under microscope.
9
Immunohistochemical Quantification of Neutrophils
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Consecutive 5-µm-thick sections were prepared from frozen brain blocks and paraffin blocks from the heads (coronary sections) and were either stained with haematoxylin and eosin (HE, Sigma-Aldrich) or subjected to immunohistochemistry for the demonstration of neutrophils as described before.32 Cryosections were fixed with acetone for 10 min at −20℃ and rehydrated in PBS for 5 min. Sections prepared from paraffin embedded heads were deparaffinized and incubated with citrate buffer (pH 6.0, 20 min at 98℃) for antigen retrieval. Incubation with the primary rat anti-Ly6G antibody (clone 1A8, Biolegend) was performed for deparaffinized sections at 4℃ for 15–18 h and for crysections at RT for 1 h, respectively. All sections were subsequently incubated with a biotinylated secondary anti-rat antibody (RT, 30 min) and thereafter with a preformed streptavidin–biotin complex (RT, 30 min; both Vectastain ABC-Kit, Vector Laboratories, USA). Visualization with diaminobenzidin or peroxidase substrate (AEC Kit, Vector Laboratories) was followed by haematoxylin (Sigma-Aldrich) counterstaining. All sections were mounted with an aqueous medium (Aquatex, Millipore). To assess the effects of α4-integrin blockade on neutrophil numbers, brain sections were inspected by a person blinded to the treatment group.
10
Histological Analysis of Rat Spinal Tissue
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Isolated rat spines and adjacent vertebral bodies were fixed in 4% paraformaldehyde, decalcified in ethylenediaminetetraacetic acid (EDTA), embedded in paraffin and sectioned at 4 μm thickness from the mid‐sagittal plane. Sections were stained with either haematoxylin and eosin (H&E; Sigma‐Aldrich, St. Louis, MO, USA) or Alcian Blue 8GX (Sigma‐Aldrich) using standard procedures and photographed at 100× magnification (Nikon Eclipse Ts100; Nikon Instruments, Melville, NY, USA).
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