The animal care and use procedures were approved by the Animal Care Committee of the University of Tokyo (Permit Number: P09-015). Male C57 BL mice were purchased from Oriental Yeast (Tokyo, Japan) and fed standard laboratory chow (CLEA Japan, Tokyo, Japan). After drug treatment for 7 days, the mice were sacrificed to extract the descending aortas. The tissues were fixed in 4% paraformaldehyde, dehydrated, and then embedded in paraffin. The paraffin-embedded tissues were cut into cross sections (2 µm). The sections were deparaffinized and rehydrated for immunohistochemistry. Tissue sections were incubated with rabbit anti-phospho eNOS (Ser1177) or -eNOS antibodies diluted at 1:200, followed by a biotinylated anti-rabbit secondary antibody and then horseradish peroxidase-labeled streptavidin, according to the manufacturer's instructions (DAKO, Copenhagen, Denmark). Staining intensities were measured in 30 randomly selected fields of the endothelium and media by Scion Image program. The intensity ratio of the endothelium to media was then compared statistically.
Horseradish peroxidase labeled streptavidin
Manufactured by Agilent Technologies
Sourced in United Kingdom
Horseradish peroxidase-labeled streptavidin is a protein complex consisting of the enzyme horseradish peroxidase covalently attached to the protein streptavidin. Streptavidin has a high affinity for the vitamin biotin, allowing it to be used as a detection reagent in various bioanalytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ab76067, by Abcam (2 mentions)
Resorcin fuchsin staining solution, by Muto Pure Chemicals (2 mentions)
Hematoxylin staining solution, by Muto Pure Chemicals (2 mentions)
Van gieson staining solution, by Muto Pure Chemicals (2 mentions)
Standard laboratory chow, by CLEA Japan (1 mentions)
Ab18147, by Abcam (1 mentions)
Ls b2138, by LifeSpan BioSciences (1 mentions)
L2515, by Merck Group (1 mentions)
5 tetramethylbenzidine liquid substrate, by Merck Group (1 mentions)
96 well microtiter plate, by Thermo Fisher Scientific (1 mentions)
L2630, by Merck Group (1 mentions)
Nunc maxisorp elisa plate, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase labeled goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Dako envision system, by Agilent Technologies (1 mentions)
6 protocols using horseradish peroxidase labeled streptavidin
1
Endothelial eNOS Phosphorylation in Mice
2
Histomorphological Analysis of Penis Tissues
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Penis tissues were fixed by perfusion with 4% paraformaldehyde and then embedded in paraffin. The specimens were sliced at a thickness of 5 μm, deparaffinized, rehydrated, and subjected to histochemical analysis. Elastica van Gieson staining was performed according to the standard method. In brief, the specimens were stained with resorcin fuchsin staining solution (Muto Pure Chemicals, CO., LTD., Tokyo, Japan), hematoxylin staining solution, and van Gieson staining solution (Muto Pure Chemicals, CO., LTD., Tokyo, Japan). For immunohistochemistry, sections were incubated with a primary antibody reactive to VE-Cad (1:400; LS-B2138; LifeSpan BioSciences), SMA (1:200; ab18147; Abcam) or nNOS (1:400; ab76067; Abcam). Sections were then incubated with biotinylated secondary antibody prior to horseradish peroxidase-labeled streptavidin according to the manufacturer's instructions (DAKO, Cambridgeshire, UK).
3
Quantifying Peptide-LPS/LTA Binding
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96-well microtiter plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with 5 µg/mL of purified LPS (from E. coli O111:B4, Sigma L2630) or LTA (from S. aureus, Sigma L2515) in PBS and then incubated for 2 h at room temperature in blocking solution (20 mM Tris-HCl pH 7.4 plus 0.05% Tween 20 and 1% BSA). Biotin-labeled peptides/proteins (2.5–20 µg/mL) were added and incubated overnight at 4°C in blocking solution. After extensive washing, bound peptides/proteins were detected by the addition of horseradish peroxidase-labeled streptavidin (1:5,000 dilution; DAKO) for 1 h at room temperature. Color was developed by adding 3,3’,5,5’-tetramethylbenzidine liquid substrate (Sigma) and optical density read at 405–620 nm.
4
Histological Analysis of Penis Structure
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The penis was fixed using perfusion with 4% paraformaldehyde. It was then embedded in paraffin and sliced into 5-μm cross sections that were deparaffinized, rehydrated, and subjected to the Elastica van Gieson stain. Elastica van Gieson stain was performed by a standard method. In brief, the specimens were consecutively stained with resorcin‐fuchsin staining solution (Muto Pure Chemicals, CO., LTD., Tokyo, Japan), hematoxylin staining solution, and van Gieson staining solution (Muto Pure Chemicals, CO., LTD., Tokyo, Japan). For immunohistochemistry, sections were incubated with a primary antibody reactive to neural nitric oxide synthase (nNOS: 1:400, Abcam, ab76067) and heterochromatin protein-1γ (HP-1γ: 1:400, Abcam, ab66617). Sections were subsequently incubated with biotinylated secondary antibody and finally horseradish peroxidase-labeled streptavidin according to the instructions provided by the manufacturer (DAKO, Cambridgeshire, UK).
5
ELISA Protocol for Peanut-Specific Antibodies
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Ninety-six-well Nunc Maxisorp ELISA plates (Thermo Fisher Scientific) were coated with 1 mg/mL in carbonate buffer at 48C overnight. After blocking with PBS/0.15% casein solution for 2 hours, plates were washed 5 times with PBS/0.05% Tween. Serial dilutions of sera were added to the plates and incubated for 2 hours at 48C. Plates were then washed 5 times with PBS/0.05% Tween. Thereafter, horseradish peroxidase-labeled goat anti-mouse IgG (The Jackson Laboratory, Bar Harbor, Me) antibodies were incubated at 48C for 1 hour. For determination of peanut extract specific IgG subclasses, biotin-labeled mouse anti-mouse IgG 1 (The Jackson Laboratory), biotin-labeled mouse antimouse IgG 2a (Becton Dickinson), or biotin-labeled rat anti-mouse IgG 2b (Bio-Legend) were used as detection antibodies for 1 hour at 48C. Thereafter, horseradish peroxidase-labeled streptavidin (DakoCytomation, Copenhagen, Denmark) was incubated at 48C for 1 hour. ELISAs were developed with 3,30,5,50-tetramethyl-benzidine and H 2 O 2 and stopped with 1 mol/L sulfuric acid. ODs were measured at 450 nm. Half-maximal antibody titers are defined as the reciprocal of the dilution leading to half of the OD measured at saturation.
6
Immunohistochemical Analysis of Canine Colorectal Tissue
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After being subjected to deparaffinization and antigen retrieval (Suppl. Table 2), the tissue sections were treated with 3% hydrogen peroxide in methanol at room temperature for 5 minutes and then incubated with 8% skimmed milk in Trisbuffered saline at 37 C for 40 minutes to block nonspecific reactions. Subsequently, the sections were incubated overnight with primary antibodies (Suppl. Table 2) at 4 C. After being washed in Tris-buffered saline, the sections were incubated with horseradish peroxidase-labeled polymer (Dako Envisionþ System; Dako, Carpinteria, CA, USA) or biotinylated anti-goat immunoglobulin G (KPL, Guildford, UK) before being incubated with horseradish peroxidase-labeled streptavidin (Dako). Finally, the reaction products were visualized with 0.05% 3-3 0diaminobenzidine and 0.03% hydrogen peroxide in Tris/HCl buffer and then counterstained with Mayer's hematoxylin. Colorectal tissue samples from dogs without lesions were used as positive controls. The primary antibodies were omitted to produce negative controls of normal colorectum, inflammatory polyp, adenoma, and adenocarcinoma.
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