Vk*MYC mouse bones were formalin fixed and decalcified in Fix Decal (Pro-Eco) overnight. Bones were then paraffin-embedded, and 4-μm sectioned. Sections were stained with primary antibodies: anti-CD31 rabbit mAb (Neomarkers, Fremont, CA, USA; diluted 1:200 over-night) or with IRF4/MUM1 mouse mAb (Santa Cruz Biotechnology, CA, USA, diluted 1:500 for 1 h). Samples were washed and incubated with biotinylated horseradish peroxidase (HRP)-labeled secondary antibodies used 1:2 for 30 min (BioCare, Birmingham, UK). Slides were then incubated for 5 min with the chromogen 3,3′ – diaminobenzidine (DAB), which is converted in an insoluble brown precipitate by the HRP. After washing, slides were contrasted with Mayer-Hematoxylin (BioOptica, Milan, IT) and mounted with a cover glass. All sections were blindly evaluated by an expert hematopathologist.
Mayer hematoxylin
Manufactured by Bio-Optica
Mayer hematoxylin is a laboratory reagent used in histology and cytology for the staining of nuclei. It is a dye solution that specifically binds to the DNA and RNA present in cell nuclei, allowing them to be visualized under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Refrigerated microtome, by Leica (2 mentions)
Axioscop2 mot plus microscope, by Zeiss (2 mentions)
Anti cd3, by Agilent Technologies (1 mentions)
Anti mica b, by R&D Systems (1 mentions)
Dakocytomation liquid dab substrate chromogen system, by Agilent Technologies (1 mentions)
Tween 20, by Merck Group (1 mentions)
Bx60 microscope, by Olympus (1 mentions)
Neutral formalin solution, by Merck Group (1 mentions)
Eukitt, by Bio-Optica (1 mentions)
Masson trichrome staining, by Merck Group (1 mentions)
Accustain trichrome stain kit, by Merck Group (1 mentions)
4 protocols using mayer hematoxylin
1
Immunohistochemical Analysis of Vk*MYC Mouse Bones
2
Immunohistochemical Analysis of CAR+CIK, NKG2D Ligands, and CD44v6 in Explanted Tumors
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In selected cases (2 mice per group) we explored by IHC the presence of CAR+CIK in the explanted tumors along with the expression of NKG2D ligands (MIC A/B; ULBPs 2,5,6) and CD44v6.
Sections from formalin fixed, paraffin-embedded samples were cut into 3-μm thick sections. The tissue slides were treated according to standard immunohistochemistry procedures. In short, the slides were permeabilized in 0.1% Triton X-100 and 0.3% Tween 20 (Sigma-Aldrich) in TBS, treated for 30 min with 1% hydrogen peroxide to quench endogenous peroxidases. The slides were incubated with individual primary antibodies overnight at 4°C inside a moist chamber. After rinsing in PBS, a secondary antibody was added. Secondary HRP-conjugate antibodies (EnVision; DakoCytomation) were used for immunohistochemistry and the reaction was visualized with DAB chromogen (DakoCytomation Liquid DAB Substrate Chromogen System, Dako). The tissues were counterstained with Mayer hematoxylin (Bio-Optica), mounted on glass slides and visualized with a BX-60 microscope (Olympus) equipped with a color Qicam Fast 1394-digital CCD camera (12 bit; QImaging). The tissues were stained with the following primary antibodies: anti–CD3 (DAKO); anti-MIC A/B (clone #159207, R&D SYSTEM, BIOTECHNE BRAND); anti-ULBP 2/5/6 (R&D SYSTEM, BIOTECHNE BRAND); anti-CD44v6 (clone #SP37, Acris, an OriGene Company).
Sections from formalin fixed, paraffin-embedded samples were cut into 3-μm thick sections. The tissue slides were treated according to standard immunohistochemistry procedures. In short, the slides were permeabilized in 0.1% Triton X-100 and 0.3% Tween 20 (Sigma-Aldrich) in TBS, treated for 30 min with 1% hydrogen peroxide to quench endogenous peroxidases. The slides were incubated with individual primary antibodies overnight at 4°C inside a moist chamber. After rinsing in PBS, a secondary antibody was added. Secondary HRP-conjugate antibodies (EnVision; DakoCytomation) were used for immunohistochemistry and the reaction was visualized with DAB chromogen (DakoCytomation Liquid DAB Substrate Chromogen System, Dako). The tissues were counterstained with Mayer hematoxylin (Bio-Optica), mounted on glass slides and visualized with a BX-60 microscope (Olympus) equipped with a color Qicam Fast 1394-digital CCD camera (12 bit; QImaging). The tissues were stained with the following primary antibodies: anti–CD3 (DAKO); anti-MIC A/B (clone #159207, R&D SYSTEM, BIOTECHNE BRAND); anti-ULBP 2/5/6 (R&D SYSTEM, BIOTECHNE BRAND); anti-CD44v6 (clone #SP37, Acris, an OriGene Company).
3
Liver Tissue Lipid Quantification
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The left lobe of the liver was fixed in 10% neutral formalin solution (Sigma-Aldrich) overnight at 4 °C, cryopreserved in a 30% (w/v) sucrose solution for 24 h at 4 °C, and stored at −80 °C. Seven μm-thick liver sections were cut with a refrigerated microtome (Leica), collected on poly-L-lysine–coated glass slides, and stored at −80 °C until staining. Oil Red O lipid stain was done with a 5% solution of Oil Red in Propylene glycol for 10 min, in a heater, at 60 °C, followed by a 5 min of wash in 85% Propylene glycol solution (Oil Red O and Propylene glycol - Sigma). Hematoxylin–eosin (H&E) counterstaining was done on frozen slides with Mayer hematoxylin (Bio-Optica) for 1 min and, after water rinsing, with 1% eosin aqueous solution (Bio-Optica) for 4 min. After staining, the slides were cleared in xylenes and cover slipped with xylenes-based mounting medium (Eukitt, Bio-Optica). The liver sections were evaluated in blind by light microscopy. Images of the stained sections were captured using Microscope Axioscop2 mot plus (Zeiss). Quantitative analysis of lipid droplet-stored triglycerides was done using ImageJ imaging software55 (link).
4
Histological Analysis of Liver Tissue
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The left lobe of the liver was fixed in 10% neutral formalin solution (Sigma–Aldrich) overnight at 4 °C, cryopreserved in a 30% (w/v) sucrose solution for 24 h at 4 °C, and stored at −80 °C. Liver sections 7-μm thick were cut with a refrigerated microtome (Leica), collected on slides, and stored at −80 °C until staining. Hematoxylin–eosin (H&E) staining was performed on the frozen slides with Mayer hematoxylin (Bio-Optica) for 1 min and, after washing with water, with 1% eosin aqueous solution (Bio-Optica) for 4 min. Oil Red O staining was performed as previously described [1 (link)]. The Accustain Trichrome stain kit was used for Masson trichrome staining (Sigma–Aldrich). After staining, the slides were cleared in xylenes and cover slipped with xylene-based mounting medium (Eukitt, Bio-Optica). The liver sections were evaluated in a blinded fashion under a light microscope. Semi-quantitative grading of the hepatocellular vacuolar degeneration and portal inflammation (i.e., the infiltration of mononuclear inflammatory cells) was also performed in a blinded fashion. Images of the stained sections were captured using Microscope Axioscop2 mot plus (Zeiss).
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