We incubated the lung tissues overnight in 10% buffered formalin. Four-micron sections were cut from paraffin embedded tissue blocks and deparaffinized in zylene and rehydrated in graded alcohols (100%-70%). Heat antigen retrieval was obtained by boiling of tissue sections in antigen retrieval solution pH 6.0 or pH 9.0 (Dako, Carpinteria, CA, USA) for 10 min in microwave prior to incubation at 4℃ overnight with primary antibodies against ET-1 (SantaCruz biotechnology, Santacruz, CA, USA), ERA (SantaCruz biotechnology, Santacruz, CA, USA), eNOS (SantaCruz biotechnology, Santacruz, CA, USA) and MMP-2 (Abcam, Campridge, UK). After incubation with the optimal biotinylated secondary antibodies for 30 min at 4℃ and afterwards with a streptavidin (Dako, Kyoto, Japan), color development was done using 3-amino-9-ethylcarbazole or DAB as a chromogen.
Streptavidin
Manufactured by Agilent Technologies
Sourced in Japan, United States
Streptavidin is a tetrameric protein derived from the bacterium Streptomyces avidinii. It has a high affinity for the vitamin biotin, forming a stable non-covalent complex. Streptavidin is commonly used in various biotechnological and biomedical applications as a tool for protein purification, detection, and immobilization.
Automatically generated - may contain errors
Lab products found in correlation
Spiral chamber, by Abnova (3 mentions)
Fluorescent microscope, by Keyence (3 mentions)
Antigen retrieval solution, by Agilent Technologies (2 mentions)
Calcein green am, by Thermo Fisher Scientific (2 mentions)
Cmtpx red cell tracker, by Thermo Fisher Scientific (2 mentions)
Bz x700 analysis software, by Keyence (2 mentions)
Normal goat serum (ngs), by Agilent Technologies (1 mentions)
Anti cd3, by Agilent Technologies (1 mentions)
Ncl dys2, by Leica Biosystems (1 mentions)
Ncl mhcd, by Leica Biosystems (1 mentions)
Ncl dysb, by Leica Biosystems (1 mentions)
Ncl g sarc, by Leica Biosystems (1 mentions)
Ncl drp2, by Leica Biosystems (1 mentions)
Optical microscopy, by Nikon (1 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
Bcl2 associated x (bax), by Proteintech (1 mentions)
Bcl 2, by Proteintech (1 mentions)
Timp 1, by Abcam (1 mentions)
Hematoxylin eosin, by Merck Group (1 mentions)
Ba 7000, by Vector Laboratories (1 mentions)
17 protocols using streptavidin
1
Immunohistochemical Analysis of Lung Tissue
2
Immunohistochemical Analysis of Cellular Markers
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We sliced formalin-fixed 4-µm section from paraffin embedded tissue blocks and then deparaffinizing with zylene and rehydrating by serial dilutions of alcohol (70%–100%) were done. Heat antigen retrieval was achieved at 100℃ for 10 minutes in microwave before incubation at 4℃ overnight.
Primary antibodies were used for B-cell lymphoma-2 (Bcl-2)-associated X (Bax), caspase-3, Bcl-2, TNF-α, IL-6, matrix metalloproteinase (MMP)-2, endothelial nitric oxide synthase (eNOS), endothelin receptor A (ERA) from Santacruz Biotechnology, Santa Cruz (Santa Cruz, CA, USA) and endothelin (ET)-1 from Abcam (Cambridge, UK). Slides were incubated with the biotinylated secondary antibodies for 30 minutes at 4℃ and then with a streptavidin (Dako, Kyoto, Japan). Color development was accomplished using 3-amino-9-ethylcarbazole or DAB as a chromogen. Densities were evaluated by using Image J and expressed in arbitrary units.
Primary antibodies were used for B-cell lymphoma-2 (Bcl-2)-associated X (Bax), caspase-3, Bcl-2, TNF-α, IL-6, matrix metalloproteinase (MMP)-2, endothelial nitric oxide synthase (eNOS), endothelin receptor A (ERA) from Santacruz Biotechnology, Santa Cruz (Santa Cruz, CA, USA) and endothelin (ET)-1 from Abcam (Cambridge, UK). Slides were incubated with the biotinylated secondary antibodies for 30 minutes at 4℃ and then with a streptavidin (Dako, Kyoto, Japan). Color development was accomplished using 3-amino-9-ethylcarbazole or DAB as a chromogen. Densities were evaluated by using Image J and expressed in arbitrary units.
3
Immunohistochemical Analysis of Muscle Proteins
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Immunohistochemical analysis involved the use of immunoperoxidase techniques on frozen sections. MANDYS110 clone 3H10, provided by the Wolfson Centre for Inherited Neuromuscular Disease, NCL-DYSB and NCL-DYS2 (Novocastra Laboratories, Newcastle on Tyne, UK) mouse monoclonal antibodies were used for dystrophin protein detection (1∶50 for each ones). Other primary antibodies used were IVD31A9 (1∶50, Developmental Studies Hybridoma Bank, Iowa City, IA), NCL-g-Sarc (1∶10, Novocastra Laboratories) and NCL-DRP2 (1∶60, Novocastra Laboratories) for alpha-sarcoglycan, gamma-sarcoglycan, and utrophin detection respectively, NCL-MHCd (1∶20, Novocastra Laboratories) for developmental myosin heavy chain isoform, anti-complement 5b-9 (1∶250, Calbiochem, Strasbourg, France) and anti-CD3 (1∶100, Dako, Glostrup, Denmark) for lymphocytes. Briefly, transverse cryosections were incubated in PBS with 5% normal goat serum (Dako) for 1 hour at room temperature. They were then incubated with primary antibody in 5% rat serum overnight at 4°C and with biotinylated secondary antibodies (E433, 1∶300, Dako) in PBS with 5% rat serum for 1 hour. Bound antibodies were detected either with streptavidin (P397; Dako) and DAB Liquid Substrate (Dako) for immunoperoxidase. Fiber type was determined using histochemical myosin-ATPase reaction after preincubation at pH 4.2, 4.35, and 10.4 as previously described [22] .
4
Immunohistochemical Analysis of Nek2 in Tissues
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IHC experiments were conducted on 5 µm formalin-fixed, paraffin-embedded sections of healthy tissue and tumor samples. The tissue sections were mounted on slides, treated with xylene, and rehydrated using alcohol-water mixtures before heat treatment in 10 mM citric acid (pH 6.0) for antigen retrieval. Endogenous peroxidase activity was stopped by the use of 0.3% H2O2 solution. After blocking with 10% bovine serum albumin (Beyotime Institute of Biotechnology, Haimen, China; ST023) the sections were incubated at 4°C overnight with primary human Nek2 antibodies (Abcam, Cambridge, UK; Ab55550; 1:400 dilution), rinsed, and incubated with biotinylated anti-mouse IgG (Dako North America Inc., Carpinteria, CA, USA; F0232; 1:100 dilution) for 30 min at room temperature. After rinsing, the samples were treated with streptavidin and biotinylated horseradish peroxidase (Dako North America Inc.). Counterstaining with hematoxylin was performed, and the sections were dehydrated in ethanol before mounting. The sections were analyzed using optical microscopy (Nikon Corp., Tokyo, Japan; magnification, ×100 or ×400). Staining specificity was confirmed using positive (gastric mucosa) and negative (heart muscle tissue) controls. Low Nek2 expression was defined as <10% and high expression as >10%.
5
Cardiac Histopathology and Apoptosis Markers
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Rats underwent a left thoracotomy under anesthesia (chloral hydrate i.p.), and heart sections were fixed with 4% paraformaldehyde (PFA) and treated with masson-trichrome staining for histopathological examination. Heart sections was made into paraffin sections. After general dewaxing and antigen reparation, the tissue slices were overlayed with primary antibodies, including B-cell lymphoma-2 (Bcl-2, Proteintech, China) and B-cell associated X (BAX, Proteintech, China) at 4°C overnight. Slides were incubated with the biotinylated secondary antibodies for 30 min at 4°C and then with a streptavidin (Dako, Kyoto, Japan). Color development was accomplished using DAB as a chromogen.
6
Immunohistochemical Detection of Endothelin-1
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Excised lung tissues were incubated overnight in 10% buffered formalin. Four-micrometer-thick sections were cut from paraffin embedded tissue blocks, deparaffinized in xylene, and rehydrated in graded alcohol solutions (70%–100%). Heat antigen retrieval was achieved by boiling the tissue sections in antigen retrieval solution in pH 6.0 or pH 9.0 (Dako, Carpinteria, CA, USA) for 10 minutes in a microwave prior to incubation at 4℃ overnight with primary antibodies against endothelin-1 (ET-1; Abcam, Cambridge, MA, USA). After incubation with the appropriate biotinylated secondary antibodies for 30 minutes at 4℃ and subsequently with streptavidin (Dako, Kyoto, Japan), color development was done using diaminobenzidine (DAB) as a chromogen and counterstained with hematoxylin.
7
Immunohistochemical Analysis of Lung Tissue
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Excised lung tissues were incubated overnight in 10% buffered formalin. Four-micrometer-thick sections were cut from paraffin embedded tissue blocks, deparaffinized in xylene, and rehydrated in graded alcohol solutions (70%–100%). Heat antigen retrieval was achieved by boiling the tissue sections in antigen retrieval solution in pH 6.0 or pH 9.0 (Dako, Carpinteria, CA, USA) for 10 minutes in a microwave prior to incubation at 4°C overnight with primary antibodies against interleukin 1α (IL-1α), chemokine (C-C motif) ligand 5 (CCL5), and tissue inhibitor of metalloproteinase 1 (TIMP-1; Abcam, Cambridge, MA, USA). After incubation with the appropriate biotinylated secondary antibodies for 30 minutes at 4°C and subsequently with streptavidin (Dako, Kyoto, Japan), color development was done using diaminobenzidine (DAB) as a chromogen and counterstained with hematoxylin.
8
Quantifying Lung Inflammation Markers
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The lung lobe was sectioned into 3 portions (3–4 mm) and embedded in paraffin. Sections were stained with hematoxylin–eosin (Sigma-Aldrich, St Louis, MO, USA) using the standard protocol. To quantify neutrophils and macrophages, lung sections were respectively immunolabeled with rat monoclonal anti-myeloperoxidase (#71674; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:400) and rat monoclonal anti-F4/80 (Serotec Inc., Raleigh, NC, USA; 1:400) antibodies. We followed the protocol of Romana-Souza et al. [38 (link)] briefly, for antigen retrieval, sections were incubated with citrate buffer (pH 6.0) before labeling then endogenous peroxidase was inhibited, sections were incubated for 45 min at 24 °C in a moisty chamber primary antibody and after washing, revelation was performed using anti-rat secondary antibody followed by incubation with streptavidin (DAKO, Carpinteria, CA, USA) and diaminobenzidine was used as the chromogen. Sections were counterstained with hematoxylin. No labeling was observed on sections where the primary antibody was omitted. To quantify the number of immunostained cells, ten random fields per animal (14,689 μm2) were analyzed as described by Romana-Souza et al. [38 (link)].
9
Influenza A H1N1 ELISA Protocol
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Briefly, 96-well plates (Nunc, 96 F MAXISORP, Roskilde, Denmark) where coated with formalin-inactivated Influenza A virus H1N1 (SBL Influenza Vaccine, Sanoil Pasteur, Lyon, France) diluted in coating buffer (0.05 M sodium carbonate buffer, pH 9.5–9.7) at 5 μg/mL and incubated at +4 °C over night. Wells were washed x3 (0.9% NaCl and 0.05% Tween-20) and blocked with 3% BSA in PBS buffer for 1 hour at 37 °C. Serum samples were diluted 1:100 and further in two-fold dilutions in dilution buffer (PBS containing 0.5% BSA and 0.05% Tween-20), and incubated for 90 min at 37 °C. Plates were then washed x5 and incubated for 60 min at 37 °C with secondary biotinylated goat-anti guinea pig antibody (Vector, BA-7000) and horseradish-peroxidase (HRP) conjugated Streptavidin (DAKO, Denmark, P0397), both at a dilution of 1:3000. Plates were then washed x5 and 100 μL of tetramethyl benzidine (TMB) substrate (Sigma Aldrich, T-0440-16) was added to each well, the reaction developed for 10 min and stopped by addition of 100 μL of 2 M H2SO4. Absorbance was measured at 450 nm in an ELISA reader (VersaMax, Molecular Devices). Cut off values were calculated as the average value of negative controls OD and 2 times the SD.
10
Immunohistochemical Analysis of Autophagy Markers
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IHC staining of paraffin section slides and tissue array with polyclonal anti-Beclin-1 (Abcam), anti-p62 (Medical and Biological Laboratories), anti-FoxO1 (Cell Signaling, C29H4), anti-Cyclin D1 (Abcam), or anti- LEF-1 (Lymphoid Enhancer Binding Factor 1) (Abcam, ab22884) were performed as previously described (18 (link)). Briefly, slides were labeled with biotin-linked secondary antibody followed by Streptavidin (Dako Cytomation, Carpinteria, USA) treatment for 10 min at room temperature. The slides were treated with AEC solution for 10 min and then counterstained with 10% hematoxylin (Muto Pure Chemicals, Tokyo, Japan) and mounted with glycerol gelatin (Sigma).
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