Cultured porcine SMCs were washed twice with phosphate-buffered saline (PBS; Corning, New York, NY, USA) and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min. Subsequent to incubation with primary (anti-Cx40, anti-Cx43, anti-S100A4 and anti-α-smooth muscle actin (SMA); Abcam, Cambridge, MA, USA) and polymer helper and poly-peroxidase anti-mouse/rabbit immunoglobulin G secondary antibodies (PV-9000 kit; GBI Labs, Mukilteo, WA USA) were added for 1 h, and diaminobenzidine (Roche Diagnostics GmbH) was used for signal detection. The slides were washed under running water and tissue samples were counterstained with hematoxylin. Sections were observed and imaged using an Olympus CX31 microscope (Olympus Corporation, Tokyo, Japan).
Diaminobenzidine
Manufactured by Roche
Sourced in United States, Germany
Diaminobenzidine is a chromogenic substrate used in immunohistochemistry and other laboratory techniques. It is used to detect the presence of specific target proteins or antigens in biological samples. When exposed to the appropriate enzyme, diaminobenzidine produces a brown-colored precipitate, which can be visualized under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Envysion, by Agilent Technologies (2 mentions)
Anti cx43, by Abcam (1 mentions)
Anti s100a4, by Abcam (1 mentions)
Anti cx40, by Abcam (1 mentions)
Anti α smooth muscle actin, by Abcam (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Corning (1 mentions)
Cx31 microscope, by Olympus (1 mentions)
Tbs t, by Abcam (1 mentions)
Horseradish peroxidase hrp conjugated anti rabbit igg antibody, by Merck Group (1 mentions)
Snail, by Merck Group (1 mentions)
Foxa1, by Abcam (1 mentions)
Hdac3, by GeneTex (1 mentions)
Diaminobenzidine, by Agilent Technologies (1 mentions)
Streptavidin peroxidase, by Agilent Technologies (1 mentions)
Rabbit anti ki67 antibody, by Abcam (1 mentions)
Tunel kit, by Roche (1 mentions)
Immpress anti rabbit igg, by Vector Laboratories (1 mentions)
Ab76416, by Abcam (1 mentions)
Ab6789, by Abcam (1 mentions)
16 protocols using diaminobenzidine
1
Immunohistochemical Analysis of Porcine SMCs
2
Western Blot Analysis of Transfected Cells
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Transfected or infected cells were recovered by scraping, washed twice in PBS and lysed for 30 min on ice with Tris-NaCl lysis buffer (0.01 M Tris–HCl, 0.1 M NaCl, 2 mM PMSF, and 1% NP-40), 48 hr post transfection. The lysates were centrifuged (800 g), and the protein was quantitated by Bradford assay and was separated with SDS page. Samples were prepared by heating to 90 °C in SDS sample buffer for 5 min. Proteins were resolved by 12% SDS-PAGE and transferred to nitrocellulose. The membrane was blocked in 3% non-fat skimmed milk in TBS plus 0.05% Tween 20 (TBS-T) for 2 hr at room temperature (Abcam, UK). To detect the antigen-antibody complex, the membrane was washed and then incubated with Horseradish Peroxidase (HRP) conjugated anti-rabbit IgG antibody diluted 1:10,000 (Sigma, USA) for 1 hr at room temperature, washed again and followed by developing with diaminobenzidine (Roche, Germany).
3
Quantifying Lung Tissue Protein Expression
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The expression levels of Carbonic anhydrase IX (CAIX), HDAC3, Snail, and FOXA1 were evaluated by immunohistochemistry. Sections with a 6-μm thickness were prepared by slicing lung tissue blocks embedded in paraffin. Slides were deparaffinized in xylene and rehydrated through a series of alcohols in water. Antigen retrieval was performed by incubation in citrate buffer (pH 6) in a microwave for 20 min. Endogenous peroxidase activity was blocked by adding 3% H2O2 for 10 min. After the samples were incubated overnight with primary antibodies against CAIX (Novus), HDAC3 (GeneTex, Irvine, CA, USA), Snail (Sigma–Aldrich), and FOXA1 (Abcam) at 4 °C, the samples were rinsed and incubated with secondary antibodies. Diaminobenzidine (Roche, Germany) was added, and the slides were incubated in the dark for 10 min. Expression levels were quantified using ImageJ software.
4
Quantifying Tumor Cell Proliferation and Apoptosis
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Mouse tumor tissues were sectioned, de-paraffinized and rehydrated. After antigen retrieval, tumor tissue sections were incubated with rabbit anti-Ki67 antibody (Abcam, Cambridge, MA) overnight, followed by incubation with biotinylated anti-rabbit antibody, streptavidin peroxidase and diaminobenzidine (Dako, Denmark). Tumor tissues were also subjected to TUNEL staining using the TUNEL kit (Roche, Basel, Switzerland), and visualized with diaminobenzidine. Tumor sections were counterstained with haematoxylin. Cells positively stained with the anti-Ki67 antibody or TUNEL were quantified as described previously [27 ].
5
PMSG and hCG-Induced Ovarian Tissue Analysis
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PMSG (15 IU/0.15 ml) was administered to rats on day 25 after birth, and hCG was additionally administered on day 27. The ovaries were collected 48 h after PMSG and 24 h after hCG
administration. The ovaries were fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) at 4°C overnight, and then further incubated with 0.1 M PBS at 4°C overnight.
Dehydration and paraffin embedding were performed per standard procedures. Sections of 3-µm thickness were obtained and dried overnight in an incubator at 37°C. After dewaxing, sections were
soaked in 1% hydrogen peroxide in methanol for 20 min to suppress internal peroxidase, and were washed with ABB (150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 0.25% gelatin, 0.05%
Nonidet P-40, 50 mM Tris, pH 7.4). The sections were then incubated in 2.5% normal horse serum for 1 h at room temperature (25°C). Sections were incubated overnight at 4°C with a 1:10,000
dilution of rabbit polyclonal antiserum against ANXA5 (previously developed in our laboratory [14 (link)]), and then washed with ABB followed by incubation
with the secondary antibody (ImmPRESS anti-rabbit IgG, Vector Laboratories, Burlingame, CA, USA) for 2 h. The specimens were washed again with ABB, visualized with diaminobenzidine (Roche,
Mannheim, Germany), and counter-stained with hematoxylin. Finally, the samples were dehydrated and mounted on coverslips.
administration. The ovaries were fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) at 4°C overnight, and then further incubated with 0.1 M PBS at 4°C overnight.
Dehydration and paraffin embedding were performed per standard procedures. Sections of 3-µm thickness were obtained and dried overnight in an incubator at 37°C. After dewaxing, sections were
soaked in 1% hydrogen peroxide in methanol for 20 min to suppress internal peroxidase, and were washed with ABB (150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 0.25% gelatin, 0.05%
Nonidet P-40, 50 mM Tris, pH 7.4). The sections were then incubated in 2.5% normal horse serum for 1 h at room temperature (25°C). Sections were incubated overnight at 4°C with a 1:10,000
dilution of rabbit polyclonal antiserum against ANXA5 (previously developed in our laboratory [14 (link)]), and then washed with ABB followed by incubation
with the secondary antibody (ImmPRESS anti-rabbit IgG, Vector Laboratories, Burlingame, CA, USA) for 2 h. The specimens were washed again with ABB, visualized with diaminobenzidine (Roche,
Mannheim, Germany), and counter-stained with hematoxylin. Finally, the samples were dehydrated and mounted on coverslips.
6
Immunohistochemical Analysis of Tumor Markers
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The isolated tumor tissues were fixed with 4% paraformaldehyde for 1 d, embedded in paraffin, and sliced at 4 μm. The sections were separated with xylene, followed by rehydration using gradient ethanol. Then, antigen extraction was performed with 10 mM citrate buffer. The tissue sections were then incubated in 3% H2O2 for 10 min and sealed with normal goat serum blocking solution for 20 min at room temperature. The sections were incubated with the primary antibodies to BARX2 (1:200, MA1-27,164, Thermo Fisher scientific), Ki67 (1:500, ab15580, Abcam), KRT16 (1:500, ab76416, Abcam), p-MEK1 (1:1000, ab129431, Abcam), p-ERK1/2 (1:1000, ab201015, Abcam) overnight at 4°C and with HRP-coupled secondary antibody goat anti-rabbit IgG H&L (1:2,000, ab205718, Abcam) or goat anti-mouse IgG H&L (1:2,000, ab6789, Abcam) for 60 min at ambient temperature. After that, the sections were stained with the peroxidase substrate diaminobenzidine (Roche Diagnostics, Indianapolis, IN, USA) for 20 min and counter-stained with hematoxylin for 1 min. The sections were paraffin-embedded after routine dehydration. Staining was observed and captured with a Leica microscope (Leica, Bannockburn, IL, USA), and brownish granules were regarded as positive expression. Analysis was performed on ImageJ, and gene expression was measured by optical density value.
7
Cav1 Immunohistochemistry Staining
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Immunohistochemistry was performed in all cases. Three micrometer-thick serial paraffin sections for each case were processed by immunohistochemistry using an antibody against Cav1 (Santa Cruz, Santa Cruz, CA, USA, rabbit polyclonal, diluted 1/350) with an automated immunostainer (Ventana BenchMark Auto-Stainer, Ventana Medical Systems). A biotin-free, dextran chain-based detection system (EnVysion, Dako, Carpinteria, CA, USA) was used for visualization, and diaminobenzidine (Ventana Medical Systems, Tucson, AZ, USA) was used as a chromogen, according to standard protocols. Vascular endothelium represented an internal positive control.
8
Immunohistochemical Analysis of Rectal Cancer
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Immunohistochemistry was performed in all but five cases of pre-treatment biopsies, which had insufficient tissue from the rectal biopsy. Resected surgical specimens with residual cancer (TRG2-5) were analyzed as well. Three ìm thick serial paraffin sections of each case were processed by immunohistochemistry using an automated immunostainer (Ventana BenchMark AutoStainer, Ventana Medical Systems, Tucson, AZ, USA) with antibodies against YKL-40 (Quidel Corporation, San Diego, CA, USA, rabbit polyclonal, diluted 1/400) and c-Met (Abcam PLC, Cambridge, UK, rabbit polyclonal, diluted 1:200). The antigen retriewal step was included in the automated programme. A biotin-free, dextran chain-based detection system (EnVysion, Dako, Carpinteria, CA, USA) and diaminobenzidine (Ventana Medical Systems, Tucson, AZ, USA) were used as the chromogen, according to standard protocols. Neoplastic tissues were assembled on tissue microarrays, excluding the primary antibody and IgG-matched serum, and were used as positive and negative controls.
9
Immunohistochemical Analysis of Tumor Tissue
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Formalin-fixed, paraffin-embedded sections (5 μm) of resected tumor tissue were dewaxed, rehydrated, and microwaved for 30 minutes in 0.01 M citrate buffer (pH 6.0) containing 0.1% Tween 20. Sections were washed in Tris-buffered saline (pH 7.6) containing 5% fetal calf serum (Life Technologies, Carlsbad, CA, USA) for 20 minutes. The primary antibodies used were: anti-Ki67 antigen antibody solution (MIB-1, M7240, Dako; 1:75 diluted with Antibody Diluent [Dako ChemMate; Dako, Glostrup, Denmark]) and anti-cleaved caspase-3 rabbit monoclonal antibody solution (Asp175, Cell Signaling Technology Inc.). The remainder of the procedure was performed on an automated immunostainer (Discovery XT, Ventana Medical Systems Inc., Oro Valley, AZ, USA). Diaminobenzidine (Ventana Medical Systems Inc.) liquid served as the chromogen.
10
Quantifying UCP1 and Adipocyte Size in Gonadal Fat
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