The skin grafts were fixed in 4% paraformaldehyde and embedded in paraffin. The blocks were sliced into 5-µm sections and stained with anti-mouse CD3ε (A0452, Dako, Glostrup, Denmark) or anti-mouse Iba-1 (019-19741, Wako, Osaka, Japan) as the primary antibody, and then with anti-rabbit IgG-horse radish peroxidase (K4003, Dako) as a secondary antibody. Antibody binding was detected with a chromogenic substrate for horseradish peroxidase. The samples were counter stained with hematoxylin and eosin or Kernechtrot (Merck Millipore, Billerica, MA, USA).
Kernechtrot
Manufactured by Merck Group
Sourced in United States, Germany
Kernechtrot is a histological stain used in microscopy. It is a red dye that selectively binds to nucleic acids, particularly DNA, and is commonly used to stain cell nuclei. The stain allows for the visualization and identification of cellular structures during microscopic examination.
Automatically generated - may contain errors
Lab products found in correlation
A0452, by Agilent Technologies (2 mentions)
K4003, by Agilent Technologies (2 mentions)
β galactosidase staining kit, by Cell Signaling Technology (2 mentions)
Mouse anti iba 1, by Fujifilm (1 mentions)
Hematoxylin and eosin, by Merck Group (1 mentions)
Bz 9000 microscope, by Keyence (1 mentions)
Lm100, by Olympus (1 mentions)
7 protocols using kernechtrot
1
Immunohistochemical Analysis of Skin Grafts
2
Histological Embryo Evaluation Protocol
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For histological evaluation, embryos were fixed in 4% paraformaldehyde in PBS. Next, 6‐μm sections of paraffin‐embedded tissue samples were stained with hematoxylin and eosin. Anti‐E‐cadherin and antivimentin antibodies were used for immunostaining. Embryo sections were incubated with the primary antibody overnight at 4 °C and then with the biotin‐conjugated secondary antibody for 2 h at room temperature. The color was developed using DAB‐Ni. Nuclei were counterstained with Kernechtrot (Merck Millipore, Darmstadt, Germany). The stained samples were photographed using a BZ‐9000 microscope (Keyence, Osaka, Japan). All antibodies are listed in Table S1 .
3
Immunohistochemical Analysis of T Cells
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The skin grafts were fixed in 4% paraformaldehyde and embedded in paraffin. The blocks were sliced into 5-μm sections and stained with anti-mouse CD3ε (A0452, Dako, Glostrup, Denmark) as the primary antibody and then with anti-rabbit IgG-horse radish peroxidase (K4003, Dako) as a secondary antibody. Antibody binding was detected with a chromogenic substrate for horseradish peroxidase. The samples were counter stained with hematoxylin and eosin (HE) or Kernechtrot (Merck Millipore, Billerica, MA, USA).
4
Laser Capture Microdissection of Carcinoma Cells
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Frozen 10-μm tissue sections were fixed with 70% ethanol for 10 min and stained with Kernechtrot (Merck, Darmstadt, Germany). A total of ~1,000 carcinoma cells in the frozen sections were punched out using a LCM system (LM100; Olympus Corporation, Tokyo, Japan) and collected on a plastic cap (CapSureTM LCM Caps; Arcturus, Mountain View, CA, USA). All specimens were assessed histologically by a pathologist (Professor E. Kawahara).
5
Cellular Senescence Assessment via β-Galactosidase
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Cellular senescence was assessed using the β-galactosidase staining kit (Cell Signaling) following the manufacturer’s protocols. Cells treated with siRNAs were cultured in 35-mm dishes for 1, 3, and 5 days. After washing with PBS, the cells were fixed and stained for β-galactosidase activity using the staining solution overnight at 37 °C, and counterstained with Kernechtrot (Sigma–Aldrich). Stained cells were observed under a microscope and photographed. The data were expressed as the percentage of β-galactosidase-positive cells.
6
Quantifying Cellular Senescence via β-Galactosidase Assay
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Cellular senescence was assessed using the β-galactosidase staining kit (Cell Signaling) following the manufacturer's protocols. Cells treated with siRNAs were cultured in 35-mm dishes for 24, 72, 96, and 120 h. After washing with PBS, the cells were fixed with 10% formalin solution for 15 min, stained for β-galactosidase activity using the staining solution overnight at 37°C, and counterstained with Kernechtrot (Sigma-Aldrich). Stained cells were viewed under a microscope and photographed. The data were expressed as the percentage of β-galactosidase-positive cells.
7
Quantification of IL4R and IL10R Expression in OA Cartilage
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Cartilage samples obtained from OA knee joints (n ¼ 8; 62 ± 5 years) and healthy shoulder joints (n ¼ 8; 53 ± 4 years) were frozen in Tissue-Tec. In general, tissue with a Mankin grade 4e6 is collected representing full thickness clearly fibrillated cartilage 39 .
Cryo-sections (6 mm) were incubated with the primary antibody overnight at 4 C (mouse anti-human IL4Ra, R&D systems or rabbit anti-human IL10Ra, LifeSpan BioSciences) (no primary antibody for the negative control). Subsequently, sections were incubated for 30 min at room temperature with the secondary antibody (Powervision Ready-to-use HRP-Anti-Mo/Ra/Rb-IgG, Immunologic). The tissue sections were counterstained with 0.1% kernechtrot (Sigma Aldrich) in 5% Al 2 (SO 4 ) 3 . The number of cells expressing IL4R or IL10R over the total number of cells at three different locations (superficial, middle, and deep zone) per section was microscopically determined. The percentage of positive cells for either IL4R or IL10R was calculated. A cell was regarded positive if a positively stained nucleus was identified in association with appropriate receptor staining.
Cryo-sections (6 mm) were incubated with the primary antibody overnight at 4 C (mouse anti-human IL4Ra, R&D systems or rabbit anti-human IL10Ra, LifeSpan BioSciences) (no primary antibody for the negative control). Subsequently, sections were incubated for 30 min at room temperature with the secondary antibody (Powervision Ready-to-use HRP-Anti-Mo/Ra/Rb-IgG, Immunologic). The tissue sections were counterstained with 0.1% kernechtrot (Sigma Aldrich) in 5% Al 2 (SO 4 ) 3 . The number of cells expressing IL4R or IL10R over the total number of cells at three different locations (superficial, middle, and deep zone) per section was microscopically determined. The percentage of positive cells for either IL4R or IL10R was calculated. A cell was regarded positive if a positively stained nucleus was identified in association with appropriate receptor staining.
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