Human coronary artery thrombi were obtained using a thrombectomy catheter (Export catheter, Medtronic, Minneapolis, MN, USA) during percutaneous coronary intervention prior to balloon angioplasty and stent deployment in AMI patients. The coronary thrombus tissues were obtained by using an aspiration catheter. The expression of S100B and MPO (EPR20257, ABCOM, USA) in the coronary artery thrombus was examined using immunohistochemistry and immunofluorescence staining. S100B and MPO antibodies were incubated overnight at 4°C with thrombus samples; the bound S100B and MPO antibodies were stained using avidin–biotin–peroxidase method (Vector Laboratories, Burlingame, CA, USA) and photographed under a microscope (ZEISS, Image A2, Germany).
Avidin biotin peroxidase method
Manufactured by Vector Laboratories
Sourced in United States
The Avidin-biotin-peroxidase method is a powerful technique used for the detection and localization of target molecules in biological samples. It utilizes the strong and specific interaction between avidin and biotin to amplify the signal, enabling sensitive and reliable detection. The method involves the use of a primary antibody, a biotinylated secondary antibody, and an avidin-biotin-peroxidase complex. This combination allows for the efficient amplification of the target signal, making it a versatile tool for various applications in research and diagnostics.
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Lab products found in correlation
Export catheter, by Medtronic (1 mentions)
A0080, by Agilent Technologies (1 mentions)
Avidin biotin blocking reagent, by Vector Laboratories (1 mentions)
C kit antibody, by Agilent Technologies (1 mentions)
Vectastain universal elite abc kit, by Vector Laboratories (1 mentions)
Triton x 100, by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Vibratome, by Leica (1 mentions)
Diaminobenzidine dab, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Serva Electrophoresis (1 mentions)
9 protocols using avidin biotin peroxidase method
1
Assessing Coronary Thrombus Biomarkers
2
Immunohistochemical Identification of Muscle Fiber Types
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For immunohistochemical (IHC) analyses, monoclonal mouse anti-myosin Skeletal fast M4276 and Skeletal slow M8421 antibodies (Abs) (Sigma Co., St. Louis, MO, USA) were utilized for demonstration of the fast and slow myosin heavy chain (MHC) isoforms, type II and type I, respectively. Polyclonal rabbit anti-human myoglobin (A0324) and anti-human fibrinogen (A0080) Abs (Dako, Glostrup, Denmark) were employed for Mb and fibrinogen. IHC visualization was achieved by the avidin-biotin-peroxidase method (Vector Laboratories, Burlingame, California, USA). Tissue sections in which the primary Abs were replaced by phosphate-buffered saline or nonimmune serum (rabbit or mouse) were used as negative controls to confirm the specificity of the test and no immunolabelling was observed44 (link).Goat and human tissues were used as positive controls. Slow MHC recognises type I fibres (slow-twitch fibres) and fast MHC recognises type IIa, IIb and IId (IIx) (in fast-twitch fibres). Immunohistochemical identification of muscle fibre types was performed on 15 different species.
3
Histological Analysis of Skeletal Tissue
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Whole‐mount skeletal preparations were established using a standard protocol described elsewhere. For histology, bones were fixed overnight in 4% paraformaldehyde at 4°C and decalcified in 0.2 M EDTA for 1–4 weeks depending on the age of the animals. Bones were embedded in paraffin, and 3 μm longitudinal sections were obtained. For immunohistochemical staining, sections were dewaxed, rehydrated, and subjected to an antigen‐retrieval procedure. Endogenous peroxidase and nonspecific binding of antibody were blocked by treating sections with 0.3% H2O2 in methanol and 5% normal sera, respectively. Sections were incubated overnight at 4°C with the antibodies listed below. The specific binding of primary antibodies was detected using a standard avidin‐biotin peroxidase method (Vector Laboratories, Burlingame, CA), the universal immunoenzyme polymer method (Nichirei, Tokyo, Japan), or a tyramid signal amplification system (DakoCytomation, Denmark), followed by visualization with DAB. In some experiments, sections were stained for tartrate‐resistant acid phosphatase (TRAP) to identify osteoclasts. The number of osterix‐positive osteoblasts or TRAP‐positive osteoclasts in the secondary spongiosa was obtained by using Image J software (http://rsbweb.nih.gov/ij/ ).
4
Immunohistochemical Analysis of Wistar Rat Brains
5
Immunohistochemical Profiling of Xenograft Tumors
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Serial sections of formalin-fixed, paraffin-embedded (FFPE) resected xenografts, 10-μm thick, were used for histopathology analyses by H&E staining. For all tumors, the histological diagnoses were confirmed under light microscopy by an experienced pathologist. Sections for immunohistochemical staining were treated twice with 0.3% Triton X-100 (Sigma-Aldrich) in PBS for 10 minutes and then in 0.3% hydrogen peroxide solution in order to block endogenous peroxidase activity. The sections were then blocked with serum followed by an Avidin-Biotin blocking reagent (Vector Laboratories; Burlingame, CA) in order to inhibit non-specific binding in the tissue. The sections were then incubated with polyclonal rabbit anti-human CD117 (c-KIT) antibody (1:50, Dako North America, Carpinteria, CA) overnight at 4°C. Sections were next incubated with biotinylated secondary antibody and ABC reagents of the Vectastain Elite Universal ABC kit according to the manufacturer’s instructions (Vector Laboratories). The secondary antibody was detected using the Avidin-Biotin-Peroxidase method with 3,3′-diaminobenzidine as the substrate (Vector Laboratories). Negative controls were performed by omitting the primary antibody and/or using isotype control antibody.
6
Immunohistochemical Analysis of PTEN, p27, and Cyclin D1 in Colorectal Cancer
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Immunohistochemical studies for PTEN, p27 and Cyclin D1 were performed on formalin-fixed, paraffin-embedded surgical sections, consisting of the 61 CRC and 20 normal tissues. The tissue sections were deparaffinized and soaked in 0.01 M sodium citrate buffer, and the cell antigens were retrieved. Subsequent to blocking non-specific binding with 10% bovine serum, the sections were incubated with anti-human mouse monoclonal antibodies against PTEN (1:100), p27 (1:100) and cyclin D1 (1:100) (all Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4°C. Anti-rabbit goat polyclonal antibodies (Vector Laboratories, Burlingame, CA, USA) were used at dilutions of 1:50. The bound antibodies were visualized using the avidin-biotin-peroxidase method (Vector Laboratories). All sections were counterstained with hematoxylin. Negative control sections were prepared using Tris-buffered saline instead of primary antibody. Sections of tonsil tissue obtained from the excision of the tonsils with known PTEN expression were used as positive controls for PTEN.
7
Immunochemical Analysis of Neurotrophins
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Three neurotrophins, neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF), were immunostained according to the protocol described in [16] . The avidin-biotin-peroxidase method (Vector Labs., Burlingame, CA, USA) was used for immunostaining. Briefly, a cycle of brain specimen was cut at a thickness of 20 µm for 10 consecutive slices, and then at a thickness of 400 µm for one slice. Total 20 cycles covering infarcted brain region of 12 mm in length were collected for immunohistochemistry (20 µm) and western blot/polymerase chain reactions (PCR) (400 µm), respectively. After the fresh frozen sections were fixated in ice-cold acetone, they were incubated for one night at 4°C with the first antibodies and were then reacted for 1 h with the biotinylated second antibodies. We used 3,3′-diaminobenzidine tetrahydrochloride and 0.02% H2O2 for tissue staining. The average optical intensity of immunoreactivity at lesion and non-lesion cortex was calculated from a total of 20 slices per rat, and eight rats at each time point were included for the analysis.
8
Immunohistochemical Localization of ALDH1A2
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Male zebra finches were perfused intracardially, and brains were cut sagittally into 40 to 60µm sections with a vibratome (Leica). Sections were heat-treated to 95°C for 30min in a 10 mM sodium citrate buffer, pH 6.0 (Fluka). Sections were then incubated with a goat anti-Human ALDH1A2 antibody (sc-22591, 1 50, Santa Cruz Biotechnology) raised against a region near the N-terminus of the human protein (human ALDH1A2, mouse RalDH2 and zebra finch zRalDH are homologous); the secondary antibody was mouse anti-goat biotinylated (1 200, Vector Laboratories). Incubations were performed overnight at 4°C with the primary and for 2 h at RT with the secondary antibody, and followed by several washes withPBS. Sections were developed with the avidin-biotin peroxidase method (Vector Laboratories) using diaminobenzidine (DAB, Sigma-Aldrich) as a substrate, and counterstained with DAPI (4'-6-Diamidino-2-phenylindole; Serva). As a specificity control, additional sections were reacted in parallel with the same antibody pre-incubated with an ALDH1A2 immunizing peptide (Santa Cruz Biotechnology), twice as concentrated as the antibody; no staining was observed (fig. S3 ).
9
Quantification and Visualization of EB1 Expression in Lung Cancer
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Transcripts were quantified by qRT-PCR as previously described (13) using the following primer pairs: EB1 (333-bp product) 5'-CTGCGTATTGTCAGTTTATG-3' (sense) and 5'-GAGGTTTCTTCGGTTTATTC-3' (antisense); COX-2 (580-bp product), 5'-CTGGCGCTCAGCCATACAGC-3' (sense) and 5'-GGCCCTCGCTTATGATCTGTC-3' (antisense); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 305-bp product), 5'-CATCTCTGCCCCCTCTGCTGA-3' (sense) and 5'-GGATGACCTTGCCCACAGCCT-3' (antisense).
Oncomine data mining. Oncomine (Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used for data analysis and visualization as previously described (14) . The expression of EB1 was compared between lung cancer and normal lung tissue extracts.
Immunohistochemistry. Human tissue microarrays were purchased from SuperBioChips (cat. no. CC5; Seoul, korea) and immunohistochemistry was performed using an anti-EB1 mouse monoclonal antibody (dilution, 1:250; cat. no. sc-47704; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, uSA) as previously described (15) . Immunostaining was performed using the avidin-biotin-peroxidase method according to the manufacturer's instructions (Vector Laboratories, Burlingame, CA, uSA). Staining intensity was scored as follows: 0, no visible staining; 1+, weak staining; 2+, moderate staining; 3+, strong staining; and 4+, very strong staining.
Oncomine data mining. Oncomine (Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used for data analysis and visualization as previously described (14) . The expression of EB1 was compared between lung cancer and normal lung tissue extracts.
Immunohistochemistry. Human tissue microarrays were purchased from SuperBioChips (cat. no. CC5; Seoul, korea) and immunohistochemistry was performed using an anti-EB1 mouse monoclonal antibody (dilution, 1:250; cat. no. sc-47704; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, uSA) as previously described (15) . Immunostaining was performed using the avidin-biotin-peroxidase method according to the manufacturer's instructions (Vector Laboratories, Burlingame, CA, uSA). Staining intensity was scored as follows: 0, no visible staining; 1+, weak staining; 2+, moderate staining; 3+, strong staining; and 4+, very strong staining.
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