Paraffin sections of epidydimal adipose tissue were prepared as described above and incubated with the anti-F4/80 (EGF-like module-containing, mucin-like, hormone receptor-like 1) antibody (GeneTex, Irvine, CA, USA). The antibodies were detected using the Polink-2 Plus HRP Anti-Rat DAB Detection kit (Golden Bridge International Inc., Mukilteo, WA, USA) according to the manufacturer’s instructions. The tissue sections were then counterstained with Mayer’s hematoxylin (ScyTek, West Logan, UT, USA). Digital images of stained sections were acquired using an Olympus IX51 inverted microscope (magnification, 200×; Olympus).
Mayer s hematoxylin
Manufactured by ScyTek Laboratories
Sourced in United States
Mayer's hematoxylin is a histological staining solution used in microscopy. It is a nuclear stain that selectively colors the nuclei of cells blue-purple. The solution contains hematoxylin, a natural dye extracted from the logwood tree, which binds to the DNA and RNA in cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Citrate buffer, by Thermo Fisher Scientific (3 mentions)
Xylene, by Merck Group (2 mentions)
Aperio digital pathology slide scanner, by Leica (2 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (2 mentions)
Ultravision hydrogen peroxide block, by Thermo Fisher Scientific (2 mentions)
Ultravision quanto detection system, by Thermo Fisher Scientific (2 mentions)
Permount, by Thermo Fisher Scientific (2 mentions)
Ultra 5 block, by ScyTek Laboratories (2 mentions)
Ix51 inverted microscope, by Olympus (1 mentions)
Anti f4 80, by GeneTex (1 mentions)
Envision dual link system hrp dab k4065, by Agilent Technologies (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Diaminobenzidine dab, by Thermo Fisher Scientific (1 mentions)
Sc 7763, by Santa Cruz Biotechnology (1 mentions)
Dab substrate kit, by Abcam (1 mentions)
Digital pathology slide scanner, by Leica (1 mentions)
Optiview detection kit, by Roche (1 mentions)
Ez prep, by Roche (1 mentions)
Automated immunostainer, by Roche (1 mentions)
Anti lgp2 antibody, by Proteintech (1 mentions)
18 protocols using mayer s hematoxylin
1
Immunohistochemical Staining of Epididymal Adipose
2
Immunohistochemical Analysis of Protein Markers
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The paraffin-embedded tissue sections were maintained at 60°C for 1 h and then de-paraffinized in xylene (Sigma–Aldrich; Merck KGaA). The tissue sections were rehydrated in graded ethanol from 95 to 75% and PBS with 0.05% Tween-20 (PBS-T). For antigen retrieval, tissue slides were boiled in 10 mM citric acid buffer with 0.05% Tween-20 (pH 6.0) for 3 min at 121°C using a pressure cooker. After air cooling, the tissue sections were incubated with peroxidase blocking reagent (RTU, EnVision™+Dual Link System-HRP (DAB+); K4065; Dako; Agilent Technologies, Inc.) for 5 min, and then blocked with goat serum for an additional 30 min. Subsequently, the tissue sections were incubated with anti-FASN (cat. no. 3180), NF-κB p65 (cat. no. 8242), p-AKT (cat. no. 4060) and phosphor-p44/42 MAPK (ERK1/2) (cat. no. 4370) antibodies (Cell Signaling Technology, Inc.) at 4°C overnight followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The sections were rinsed with PBS-T, developed in 3′,3′-diaminobenzidine (DAB) substrate chromogen (EnVision™+Dual Link System-HRP(DAB+); K4065; Dako; Agilent Technologies, Inc.), and then counterstained with Mayer’s Hematoxylin (ScyTek Laboratories). All sections were scanned on an Aperio Digital Pathology Slide Scanner (Leica Biosystems).
3
Immunohistochemical Assessment of Angiogenesis and Collagen
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To detect vascularization and collagen formation, primary antibodies anti-VEGF (Sigma V1253, St Louis, Mo , USA) and anti-Type-II collagen (Sc7763, Santa-Cruz Biotechnology, Calif, USA) were used. After dewaxing in xylene, the sections were dehydrated with ethanol. They were then incubated with 0.5% trypsin at 37 °C for 15 minutes and endogenous peroxidase activity was inhibited using hydrogen peroxide (Merck). Blocking serum was applied for 1 hour, followed by incubation with primary antibodies anti-VEGF and anti-Type-II collagen at 4 °C overnight. The sections were then treated with the anti-mouse biotin-streptavidin hydrogen peroxidase secondary antibody (85–9043 Zymed Histostain kit). Immunoreactivity was made visible using diaminobenzidine (DAB, 00–2014, Invitrogen), and counterstaining was performed using Mayer’s hematoxylin (800-729-8350, ScyTek). The sections were coated with entellan, and evaluated using a light microscopy (BX43, Olympus, Japan) by three independent researchers.17 The procedure was performed three times. The immunoreactivity was evaluated as no (0), weak (+), moderate (++), and strong (+++), and stained cells were counted for each staining degree. The H-score value was calculated using the formula: Pi (intensity of staining + 1), where Pi is the percentage of stained cells for each intensity.17
4
Organ Histology Analysis Protocol
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At the end of the experiments, samples of the major organs, including the liver, small intestine, kidneys, and spleen, were fixed in cold 4% paraformaldehyde, embedded in paraffin, sectioned at 5 μm, and stained with 3′,3′-diaminobenzidine (DAB) substrate kit (Abcam, ab64238), and counterstained with Mayer’s hematoxylin (ScyTek Laboratories, UT, USA). All sections were scanned by an Aperio digital pathology slide scanner (Leica Biosystems, Buffalo Grove, IL, USA).
5
Immunohistochemical Assessment of PD-L1 Expression
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Formalin-fixed, paraffin-embedded tissues were sectioned at a thickness of 4 μm and stained using an automated immunostainer (Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer’s protocol. The slides were dried at 60 °C for 1 h and deparaffinized at 75 °C for 4 min using EZ Prep (Ventana Medical Systems, Tucson, AZ, USA). The cells were conditioned (heat pre-treatment) at 100 °C for 64 min using a cell conditioning solution that contained Tris/borate/ethylenediaminetetraacetic acid. The anti-PD-L1 22C3 mouse monoclonal primary antibody (Agilent Technologies, Santa Clara, CA, USA) was diluted to 1:50 and applied to the sections, which were then incubated at 37 °C for 32 min. Signals were detected using an Optiview detection kit (Ventana Medical Systems, Tucson, AZ, USA) with streptavidin-biotin staining. Counterstaining was performed for 2 min at room temperature using Mayer’s hematoxylin (ScyTek, Logan, UT, USA). PD-L1 expression was defined if membranous and/or cytoplasmic staining was observed in tumor cells and tumor-associated immune cells. The combined positive score (CPS) was recorded based on the number of PD-L1-positive tumors and immune cells in relation to the total number of tumor cells. PD-L1 positivity was defined as a CPS > 1.
6
Immunohistochemical Analysis of LGP2 Expression
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Laboratory of genetics and physiology 2 protein expression in human tumor specimens and in the SK‐N‐AS xenograft tissues was assessed by immunohistochemical analysis. Immunohistochemical staining was performed as previously described.15 Paraffin sections (2 μm) cut from formalin‐fixed paraffin‐embedded block of the xenograft tissue (day 17, n = 3/each group; day 27, n = 5/each group) were incubated overnight at 55°C, and then deparaffinized in xylene and hydrated. The sections were incubated with the anti‐LGP2 antibody (1:200; Proteintech Group) for 1 hour at room temperature. Tissue sections were visualized with ImmPACT diaminobenzidine (DAB) peroxidase substrate (Vector Laboratories, Burlingame, CA, USA). Finally, sections were counterstained with Mayer's hematoxylin (ScyTek Laboratories, Logan, UT, USA) and mounted. Staining scores were defined as 2+ (strong cytoplasmic LGP2 staining, the proportion of stained cells > 60%); 1+ (faint or weak cytoplasmic LGP2 staining, 60% > the proportion of stained cells > 30%); or 0 (no staining and only nuclear LGP2 staining). All the sections were initially scored by a clinical pathologist. Because there was a significant difference in the survival possibility between these 2 groups of patients, both scores 1 and 2 as positive staining and score 0 as negative staining for the further statistical analysis.
7
Quantifying Liver Oxidative Stress via 8-OHdG Immunohistochemistry
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To detect oxidative stress in the liver, we adopted the immunohistochemical staining of 8-OHdG. The 10% paraffin-embedded tissues were cut into 3-µm thick sections. After deparaffinization and rehydration, the sections were heated in a citrate buffer (10 mM, pH 6, Thermo Fisher Scientific, Waltham, MA, USA) in a microwave for 30 min to retrieve the antigens. After washing with PBS, all sections were stained using the UltraVision Quanto Detection System HRP DAB kit (Thermo Fisher Scientific, Chino, CA, USA) and hybridized with the anti-8-OHdG antibody (JaICA, FSZ, Tokyo, Japan). Then the sections were counterstained with Mayer’s hematoxylin (ScyTek Laboratories, UT, USA), dehydrated, and mounted using a mounting medium according to the manufacturer’s instructions. The quantification data used image J to count the number of cells and calculate the percentage of dying brown cells.
8
Immunohistochemical Analysis of Liver Tissues
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For immunohistochemical analysis, formalin-fixed paraffin-embedded blocks of the mice's liver tissues were cut into 2-μm sections. After deparaffinization and rehydration, we heated sections in a citrate buffer (10 mM, pH 6, Thermo Fisher Scientific, Waltham, MA, USA) in a microwave for 30 min to retrieve the antigens. Endogenous peroxidase activity was blocked with a 3% hydrogen peroxide (UltraVision Hydrogen Peroxide Block; Thermo Fisher Scientific) for 10 min. The sections were incubated with α-SMA antibody (ab5694, abcam, JHY) for 1 h at room temperature and then visualized using HRP polymer (UltraVision Quanto Detection System; Thermo Fisher Scientific) and DAB chromogen (DAB Peroxidase Substrate Kit; Vector Laboratories, Burlingame, CA, USA). The sections were counterstained with Mayer's hematoxylin (ScyTek Laboratories, Logan, UT, USA), dehydrated, and then mounted using a mounting medium. The staining intensity of sections was measured by independent color channel of an image J analysis 27 . Each group had six to eight samples.
9
MIOX Immunostaining Protocol for Kidney
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Unstained slides from the study population were used. Staining was performed using polyclonal MIOX antibody (1:1000; Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C overnight, followed by incubation with dextran polymer conjugated with horseradish peroxidase (GBI Labs, Bothell, WA, USA) for 5 minutes at room temperature. Finally, all sections were counterstained with Mayer’s hematoxylin (ScyTek Laboratories, West Logan, UT, USA) and evaluated under light microscopy. The MIOX score was graded in a blinded fashion by a kidney pathologist. The score was expressed semiquantitatively from 1 to 4 as follows: score 1, no staining or faint staining in a few tubules; score 2, mild staining; and scores 3 and 4, moderate and strong staining, respectively; see Supplementary Methods.
10
Immunohistochemical Analysis of PTEN in Murine Liver
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For immunohistochemical analysis, formalin-fixed paraffin-embedded blocks of the mice’s liver tissue were cut into 2-µm sections. After deparaffinization and rehydration, sections were heated in a citrate buffer (10 mM, pH 6, Thermo Fisher Scientific, Waltham, MA, USA) in a microwave for 30 min to retrieve the antigens. Endogenous peroxidase activity was blocked with a 3% hydrogen peroxide (UltraVision Hydrogen Peroxide Block; Thermo Fisher Scientific) for 10 min. The sections were then incubated with a PTEN antibody (Santa Cruz, Santa Cruz, CA, USA) for 1 h at room temperature. PTEN immunoreactivity in sections was visualized using HRP polymer (UltraVision Quanto Detection System; Thermo Fisher Scientific) and DAB chromogen (DAB Peroxidase Substrate Kit; Vector Laboratories, Burlingame, CA, USA). The sections were counterstained with Mayer’s hematoxylin (ScyTek Laboratories, Logan, UT, USA), dehydrated and then mounted using a mounting medium.
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