Knee joints from mice were fixed with 4% paraformaldehyde (PFA) (Wako Pure Chemical Industries) for 2 days, decalcified with K-CX (FALMA, Tokyo, Japan) for 2 days, and embedded in paraffin. Sections were cut into 4 μm–thick sections, deparaffinized, and rehydrated; antigen retrieval was performed by incubation overnight with ethylenediaminetetraacetic acid (EDTA) (1 mM) at pH 8.0. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxidase (H2O2) in methanol for 30 minutes. After blocking with normal horse serum (Vectastain Universal Elite ABC kit; Vector Laboratories, Burlingame, CA, USA) for 30 minutes, sections were incubated with antibodies against ALK5 (sc-398; Santa Cruz Biotechnology), FOXO1 (#2880; Cell Signaling Technology) and normal rabbit IgG (AB-105-C; R&D Systems, Minneapolis, MN, USA) for 1 hour. Sections were incubated with biotinylated secondary antibodies for 30 minutes, followed by incubation with streptavidin–peroxidase complex (Vectastain Universal Elite ABC kit) for 30 minutes. Antibody complexes were visualized using the diaminobenzidine substrate system (Wako Pure Chemical Industries), and counterstained with hematoxylin. The percentage of cells positive for ALK5 and FOXO1 was determined using the BZ-II Analyzer software (Keyence, Osaka, Japan).
Diaminobenzidine substrate system
Manufactured by Fujifilm
Sourced in United States
The Diaminobenzidine substrate system is a laboratory equipment used for the detection and visualization of various biological markers in immunohistochemistry and other related techniques. The system provides a chromogenic substrate that reacts with the enzyme-labeled antibodies, producing a colored precipitate that can be observed under a microscope. This allows researchers to identify the location and distribution of the targeted molecules within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Dako envision dual link system hrp, by Agilent Technologies (2 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
Ab 105 c, by R&D Systems (1 mentions)
Bz 2 analyzer software, by Keyence (1 mentions)
Vectastain universal elite abc kit, by Vector Laboratories (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Fujifilm (1 mentions)
Bz x800 microscope, by Keyence (1 mentions)
3 protocols using diaminobenzidine substrate system
1
Immunohistochemical Analysis of ALK5 and FOXO1 in Mouse Knee Joints
2
Immunohistochemical Analysis of Soft Tissue Leiomyosarcoma
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Immunohistochemical staining was performed as described previously [9 (link)]. We used the samples of soft tissue leiomyosarcoma registered in the files of the Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. 68 samples of soft tissue leiomyosarcoma from 68 patients were prepared for immunohistochemistry. These samples had been obtained from biopsy specimens or surgically resected tumors. Samples after chemotherapy were not included in immunohistochemistry. All samples were fixed in 10% neutral buffered formalin and embedded in paraffin. After being deparaffinized in xylene and dehydrated in a graded ethanol series, sections were pretreated with 1.0 nM EDTA (Wako Pure Chemical Industries, Osaka, Japan) in a microwave oven at 100°C for 15 minutes before being incubated with anti-TUBB3 monoclonal antibodies (1 : 100, clone TUJ1, BioLegend, San Diego, CA, USA) at 4°C overnight. Samples were then incubated with Dako EnVision Dual Link System-HRP (Agilent, Santa Clara, CA, USA), visualized using the diaminobenzidine substrate system (Wako Pure Chemical Industries, Osaka, Japan), and counterstained with diluted hematoxylin.
3
Immunohistochemical Analysis of GCTB Samples
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Surgically-resected GCTB clinical samples were fixed with 4% PFA and embedded in paraffin (FFPE). Immunostaining was performed as described previously 43 (link). Briefly, antigen retrieval of deparaffinized, rehydrated sections was performed by heating in a microwave for 20 min with 10 mM citric acid at pH 6.0 (FUJIFILM Wako). After blocking with normal goat serum for 30 min at room temperature, the sections were incubated with anti-H3G34W monoclonal Abs (1:200) at 4 °C overnight 44 (link). Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxidase in methanol for 30 min at room temperature, and specimens were then incubated with Dako EnVision™+ Dual Link System-HRP (Agilent, Santa Clara, CA, USA) for 45 min at room temperature. Antibody complex was visualized using the diaminobenzidine substrate system (FUJIFILM Wako) and counterstained with hematoxylin 44 (link). Section images were obtained on a Keyence BZ-X800 microscope (Keyence Corporation, Osaka, Japan), and nuclear immunoreactivity for H3G34W was evaluated.
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