In the IHC assay, the samples were initially fixed for 15 minutes at room temperature by using PBS containing 4% paraformaldehyde. Then, the sample slides were blocked by 10% goat serum (Thermo Fisher Scientific, Waltham, MA), permeabilized at room temperature for 1 hour by using 0.1% Triton X‐100 (Sigma Aldrich, St. Louis, MO), incubated at 4 degrees for 24 hours with primary anti‐CSE antibodies (Abcam, Cambridge, MA), washed with PBS, incubated for another 2 hours at room temperature with appropriate biotin‐conjugated secondary antibodies (Abcam, Cambridge, MA), stained with DAPI (Sigma Aldrich, St. Louis, MO), counterstained with haematoxylin and finally observed underneath an Olympus IX71 microscope (Olympus, Tokyo, Japan) to evaluate the positive protein expression of CSE in the samples using Prism 8.0 software (GraphPad, San Diego, CA).
Biotin conjugated secondary antibody
Manufactured by Abcam
Sourced in United Kingdom
Biotin-conjugated secondary antibody is a laboratory reagent used to detect and visualize target proteins in various immunoassays. It consists of a secondary antibody that has been covalently linked to the small molecule biotin. This biotin label allows for sensitive detection of the target protein when used in conjunction with a streptavidin-based detection system.
Automatically generated - may contain errors
Lab products found in correlation
Prism 8, by GraphPad (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Ix71 microscope, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Nlrp1, by Abcam (1 mentions)
Nlrp3, by Abcam (1 mentions)
5 5 dithiobis 2 nitrobenzoic acid dtnb, by Merck Group (1 mentions)
Thiobarbituric acid, by Merck Group (1 mentions)
Tetramethylbenzidine, by Merck Group (1 mentions)
Mouse monoclonal anti gfp, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Lsm 5 pa laser scanning microscope, by Zeiss (1 mentions)
5 protocols using biotin conjugated secondary antibody
1
Immunohistochemical Analysis of CSE Expression
2
Immunohistochemical Analysis of NLRP Inflammasome in SCI
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Tissues from rats (n=5 in each group) were perfused and fixed with 4% paraformaldehyde 72 h after SCI. The spinal cord tissue was sliced into 10-μm sections and fixed with paraffin. The slides underwent dewaxing, hydration, and thermal antigen repair. Antigen was inactivated by 3% hydrogen peroxide-methanol for 10 min and the slides were rinsed with PBS 3 times. The samples were blocked with goat serum (50 μl) at room temperature for 20 min and rinsed with PBS 3 times. The slides were incubated with primary antibodies NLRP1 (1: 1000; Abcam), NLRP3 (1: 1000; Abcam), and ASC (1: 1000; Novus Biologicals) overnight at 4°C. After incubation, the slides were rinsed with PBS 3 times and incubated with biotin-conjugated secondary antibodies (1: 1000; Abcam) at 37°C for 30 min. Subsequently, the samples were stained with DAB solution for 10 min, re-dyed with hematoxylin for 5 min, dehydrated with gradient alcohol, soaked in xylene for 10 min, dried, and sealed with neutral resin. The expression of positive cells was observed under a microscope and captured in high-definition pictures.
3
Nitrotyrosine ELISA with Fibrinogen
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Anti-3-nitrotyrosine polyclonal antibody, biotin-conjugated secondary antibody, and Strept/HRP were obtained from Abcam (Cambridge, UK). Peroxynitrite was synthesized according to the method of Pryor et al. [17 ]. Fibrinogen for a competitive ELISA test was prepared from human blood according to the method described by Doolittle’ et al. [18 (link)]. 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman’s reagent), thiobarbituric acid, trichloroactetic acid, and Sigma OPD Fast Substrate were purchased from Sigma-Aldrich. All other reagents were of analytical grade and were provided by commercial suppliers.
4
Quantifying GFP Expression by ELISA
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GFP expression in various tissues was quantified by ELISA (8 (link)). In brief, a primary antibody (mouse monoclonal anti-GFP; Abcam) at 1:10,000 dilution in bicarbonate buffer was used to coat a 96-well plate (Nunc Maxisorb; Sigma-Aldrich, St. Louis, MO, USA) overnight. After they were washed, the plates were incubated in turn with 300 µl/well blocking buffer (1% bovine serum albumin in PBS), 200 µg protein/well of sample or standards (serial dilutions of GFP, starting at 4000 pg/well), 100 µl/well biotin-conjugated secondary antibody (1:5000 in blocking buffer; Abcam), 100 µl/well streptavidin-horseradish peroxidase (1:20,000 in blocking buffer), with every stage incubated at 37°C for 1 h. Tetramethylbenzidine (100 µl/well; Sigma-Aldrich) was incubated at room temperature for 10 min, with care taken to avoid exposure to direct light. Color development was terminated using100 µl/well of 2.5 M H2SO4, and absorbance was read at 450 nm.
5
Immunohistochemical Analysis of Mouse Xenograft Samples
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Mouse xenograft samples were fixed in 10% formalin at room temperature overnight, embedded in paraffin and cut into 3-µm sections. Endogenous peroxidase activity was blocked with 3% H2O2 at room temperature for 15 min. Antigen retrieval was performed in citrate buffer and sections were washed with PBS. Then, sections were subsequently blocked with 1% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. Next, the sections were incubated with primary antibodies against E-cadherin (1:200; cat. no. 3195; Cell Signaling Technology, Inc.) or vimentin (1:200; cat. no. ab92547; Abcam) at 4°C overnight and the biotin-conjugated secondary antibody (1:3,000; cat. no. 14708; Cell Signaling Technology, Inc.) at room temperature for 10 min. Streptavidin-peroxidase was applied for 15 min at room temperature followed by development with DAB. Then, the sections were counterstained with Mayer's Hematoxylin for 2 min at room temperature. Subsequently, the sections were dehydrated and sealed with neutral gum. Images were photographed using the LSM 5 Pa Laser Scanning Microscope (Zeiss AG).
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