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Vector dab kit

Manufactured by Vector Laboratories
Sourced in United States

The Vector DAB kit is a laboratory tool used for the detection and visualization of target proteins or molecules in biological samples. It contains the necessary reagents for a chromogenic detection method that utilizes 3,3'-Diaminobenzidine (DAB) as the chromogen.

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22 protocols using vector dab kit

1

Immunohistochemical and Immunofluorescence Analysis

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Slides were processed as previously described[14 (link), 22 (link)]. For IHC, sections were incubated with antibody specific to Synaptophysin (Sigma, 1:250) overnight at 4°C, then in goat HRP-conjugated secondary antibody (Santa Cruz Biotechnology, 1:1000). Slides were developed for 5 minutes with a Vector DAB Kit (Vector Laboratories) per manufacturer's protocol, counterstained in CAT Hematoxylin, and imaged on a Nikon Eclipse E800 upright microscope with an Olympus DP71 camera and manufacturer's software. For IF, primary antibodies against the following proteins were used: phospho-histone H3 (pHH3) (Abcam, 1:750), Ki67 (Abcam, 1:250), phospho-Akt (Ser-473), Akt and Pten (1:100, Cell Signaling) followed by Alexa Fluor 488/546/647-conjugated secondary antibodies raised in goat (Molecular Probes,1:1000). Sections were counterstained with DAPI. Imaging was performed by confocal microscopy (Leica TCS SPE/DMI4000B; Leica Application Suite software). The numbers of pHH3- and Ki67-positive cells were counted and normalized to the total number of nuclei per image.
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2

Immunohistochemical Staining of RNF2 in Hepatocellular Carcinoma

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For perform immunohistochemical (IHC) staining for RNF2, we purchased a tissue microarray slide from Servicebio (LVC-1608; Servicebio, consisting of 83 pairs of hepatocellular carcinoma tissue and adjacent normal tissues). Tumor tissue sections from nude mice were processed according to standard protocols and routinely examined by H&E staining. Firstly, the tissue sections were heated at 60 °C for 2 h, and then dewaxed with an alcohol gradient treatment. Then blocking the slides with 3% normal sheep serum (ZSbio, Beijing, China) for 1 h at room temperature. After blocking, specimens will be incubated overnight at 4 °C with primary antibody. The VECTASTAIN Elite ABC HRP kit (Vector Laboratories, Burlingame, CA, USA) and the VECTOR DAB kit (Vector Laboratories) were then used to color development according to the manufacturer’s instructions. The slides were visualized using Pannoramic Digital Slide Scanners (3DHISTECH, Budapest, Hungary).
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3

Immunostaining for Apoptosis and Autophagy

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Sections of 4% paraformaldehyde‐fixed paraffin‐embedded tissue samples, cut at 5‐µm thickness, were used for immunostaining. Staining for cleaved caspase 3 was performed on paraffin sections using a rabbit anti‐cleaved caspase 3 antibody (1:200; Cell Signaling). The secondary antibody was a biotinylated goat anti‐rabbit IgG (1:200; BA‐1000; Vector Laboratories). Staining was performed using the Vector ABC kit and the Vector DAB kit (Vector Laboratories). Staining for LC3BII was performed on paraffin sections using a rabbit polyclonal LC3B antibody (1:200; Novus Biologicals). ImmPRESS HRP Goat Anti‐Rabbit IgG Polymer Kit (Vector Laboratories) was used as a secondary antibody. Staining was performed using a DAB substrate kit (Vector Laboratories).
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4

Histological Evaluation of Mouse Xenograft Tumors

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Mouse xenograft tumors were collected at the end of treatment and immediately fixed in 10% neutral buffered formalin. Samples were subjected to embedding in paraffin and sectioning at 5 µm. The resulting sections were used for haematoxylin and eosin (H&E) and immunohistochemistry staining. The following primary antibodies were used: DHODH (1:100; Proteintech), 4HNE (5 µg/ml; Jaica), and Ki67(1:100; Proteintech). A goat anti-rabbit IgG and a goat anti-mouse IgG were used as secondary antibodies. Immunohistochemical staining was performed using a Vector DAB kit.
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5

Immunohistochemical Analysis of SLC2A1 and GPX4

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Kidneys were regularly harvested for SLC2A1 and GPX4 immunohistochemistry (IHC). Following overnight fixation in 4% paraformaldehyde, the tissues were embedded in paraffin and sectioned at a thickness of 4 μm. After subjecting the sections to antigen retrieval in a solution containing 10 mM sodium citrate, 0.05% Tween-20 at a pH of 6.0, they were heated at 95–100°C. To block endogenous peroxidase activity, the slides were treated with 3% H2O2. Additionally, 2.5% normal horse serum was applied to minimize nonspecific binding. The slides were subsequently incubated at a temperature of 4°C overnight with rabbit anti-SLC2A1 (21829-1-AP, 1:200, Proteintech), anti-GPX4 (HuaAn, Hangzhou, China) and then treated with ImmPRESS HRP polymer horse-anti-rabbit secondary antibody at room temperature for 1 hour. Negative controls were established by substituting the primary antibody with antibody diluent. The signals were visualized using the Vector® DAB kit and counterstained with hematoxylin following the washing steps. To quantify the staining, a total of 20 to 30 fields (400× magnification) were randomly chosen from each section. The percentage of positive stained area was then analyzed utilizing ImageJ software.
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6

Immunohistochemistry of G4 DNA and Proteins

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The paraffin sections were prepared as described above. The sections were de-waxed in xylenes, re-hydrated, and boiled for 10 min in 10 mM sodium citrate buffer (pH 6.0) using a microwave. Most primary antibodies, such as anti-RHAU, were treated with the sodium citrate, whereas anti-BG4 with trypsion pretreatment (25 mg Trypsin, 314 mg CaCl2.2 H2O in 100 ml 50 mM Tris, PH7.8) incubated for 20 min at 37 °C to prevent G4 DNA forming at an artificial state. After treating with 0.3% hydrogen peroxide for 10 min, these paraffin sections were incubated with blocking solution (TBST/5% normal goat serum). Then, we washed away the blocking solution, added primary antibodies, and washed the samples three times in Tris-borate-EDTA buffer (shorted for TBS). Next, biotinylated second antibodies were added to the samples, and ABC reagent (Vector ABC kit, PK-6100) was prepared conforming to the manufacturer's instructions. Then, DAB reagent (Vector DAB kit, SK-4100) was used to stain the slides. Eventually, the slides were stained with hematoxylin following the manufacturer's instructions.
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7

Histological and Immunohistochemical Analysis of Uterine Tissue

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One uterine horn per mouse was fixed overnight in 10% neutral buffered formalin, processed in increasing concentrations of ethanol, and paraffin embedded. Tissues sections (5μm) were processed for histological analysis. Sections were stained with Hematoxylin and Eosin and analyzed by two pathologists blinded to the experimental exposures. For immunohistochemistry, sections were incubated with antibody specific to phospho-histone H3 (pHH3), alpha smooth muscle actin (αSMA) and cytokeratin 8 (CK8) (S1 Table), then with goat HRP-conjugated secondary antibody (Santa Cruz Biotechnology, 1:1000). Slides were developed for 5 minutes with a Vector DAB Kit (Vector Laboratories) per manufacturer's protocol, counterstained in CAT Hematoxylin. Representative images were taken on a Nikon Eclipse E800 upright microscope with an Olympus DP71 camera and manufacturer's software. We used primary antibody against pHH3 for immunofluorescence (S1 Table) followed by goat Alexa Fluor 488—conjugated secondary antibody (Molecular Probes, 1:1000). Sections were counterstained with DAPI imaged by confocal microscopy (Leica TCS SPE/DMI4000B; Leica Application Suite software).
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8

Quantifying AGPS and AGPAT3 Expression in Gastric Specimens

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Immunohistochemical staining was performed using 5-μm-thick sections of 4% paraformaldehyde-fixed paraffin-embedded tissue samples. The following primary antibodies were used: anti-AGPAT3 antibody (1:200; DF3642; Affinity; RRID: AB_2836014), anti-4HNE monoclonal antibody (5 µg/ml; Jaica), and anti-AGPS antibody (1:700; ab236621; Abcam; RRID: AB_2921211). Goat anti-rabbit IgG and goat anti-mouse IgG were used as secondary antibodies. Immunohistochemical staining was performed using a Vector DAB kit. Notably, immunohistochemical staining was performed to examine the expression profiles of AGPS and AGPAT3 in gastric specimens as described previously; the expression profiles were evaluated and scored by two pathologists at Nanfang Hospital for the staining intensity (scale = 0–3) and staining frequency (scale = 0–4). For statistical analysis, the expression levels of the AGPS and AGPAT3 proteins were represented by an expression score ranging from 0 to 12 and calculated using the formula intensity×frequency.
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9

Tracing Descending Pathways Post-SCI

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On the 28th day after SCI, bilateral and stereotaxical injections of biotinylated-dextran amine (BDA; MW 10,000, 10%, 1 μl/site; Molecular Probes, Eugene, OR) were made into the intermediate gray matter of the T7-8 cord segments at distances of 3 – 6 mm rostral to the contusion site (for BDA, one injection/site/mm longitudinal distances) according to previously published work (Deng et al., 2013 (link)).
BDA staining was performed according to earlier reports (Fouad et al., 2001 (link)). Slides were dried in an incubator at 38°C for 1 hr and washed twice for 10 min in 50 mM Tris-buffered saline (TBS), pH 7.4, followed by two 45 min washes with TBS containing 0.5% Triton X-100. Afterwards, the slides were incubated overnight with an avidin–biotin–peroxidase complex in TBS with Triton (ABC Elite, Vector Laboratories, Burlingame, CA) according to the manufacturer’s protocol. Subsequently, a DAB reaction was performed using the Vector DAB kit (SK4100, Vector Laboratories). The reaction was monitored and stopped by extensive washing in water.
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10

Immunohistochemical Quantification of Nerve Regeneration

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Two months after the therapeutic intervention, animals were euthanized with an overdose of pentobarbital sodium (80 mg/kg) and an intracardiac perfusion with 4% paraformaldehyde was performed. The affected portions of the SC were fixed overnight and then transferred to 30% sucrose for cryoprotection. Samples were embedded in Tissue-Tek (Miles Elkhart, IN, USA), and longitudinal frozen sections (40 μm thick) were performed. Immunohistochemical staining was carried out in order to count the amount of TH+ and 5-TH+ fibers. Tissues were incubated in 0.03% hydrogen peroxide to quench endogenous peroxidase activity. Subsequently, the tissue was incubated overnight with the following primary antibodies: monoclonal goat antibody against TH (1:2000; Chemicon), or polyclonal rabbit antibody against 5-HT (1:2000; Sigma- Aldrich). Following rinsing with PBS, samples were incubated for at least 2 h with donkey IgG anti-goat IgG (1:500; Chemicon) and Sheep IgG anti rabbit IgG (1:500; Abcam) secondary biotinylated antibodies. To visualize positive fibers, samples were incubated 5 min with Vector DAB kit (Vector laboratories, CA, USA). Then, samples were evaluated and analyzed by a blinded observer that counted individual fibers using a 20X objective (Olympus DP72, Japan). The number of regenerating axons 1 mm caudal to the lesion was assessed.
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