Sections from six-month-old mice were incubated with 3% H2O2 for 30 min at room temperature. The following antibodies were used for immunohistochemistry: anti-GFAP (AB5804, Millipore, 1:400 dilution); anti-caspase-3 [cleaved Asp175] (ab52293, Abcam, 1:400 dilution); goat anti-rabbit IgG (H&L); biotin-conjugated, affinity-purified antibody (AP132B, Millipore, 1:500 dilution); and peroxidase-conjugated streptavidin (SA202, Millipore, 1:500 dilution). A standard avidin-biotin-immunoperoxidase complex method was used.
Sa202
Manufactured by Merck Group
Sourced in United States
The SA202 is a laboratory equipment product. It serves a core function in laboratory settings, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ap132b, by Merck Group (3 mentions)
Sc 253, by Santa Cruz Biotechnology (2 mentions)
Ab52293, by Abcam (1 mentions)
Ab5804, by Merck Group (1 mentions)
Tmb solution, by Merck Group (1 mentions)
Goat anti aqp4 antibody, by Santa Cruz Biotechnology (1 mentions)
Maxisorp nunc plates, by Thermo Fisher Scientific (1 mentions)
Flexstation 3, by Molecular Devices (1 mentions)
4 protocols using sa202
1
Immunohistochemistry of Oxidative Stress
2
Immunohistochemical Detection of c-Fos
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The floating sections were rinsed in 0.01 M PB saline (PBS, pH 7.4), processed for 30 min in 0.3% H2O2 in PBS, and incubated in blocking buffer (10% bovine serum in PBS) for 1 h. Then, the sections were incubated with a rabbit polyclonal antibody against c-Fos (1:6000, sc-253, Santa Cruz Biotechnology, Santa Cruz, United States) in PBS containing 1% bovine serum for 72 h at 4°C on an agitator. After rinsing in PBS, the sections were incubated with a biotinylated goat anti-rabbit IgG (1:1000, AP132B, Millipore, Temecula, CA, United States) for 48 h at 4°C and then with horseradish peroxidase-conjugated streptavidin (1:2000, SA202, Millipore, Temecula, CA, United States) for 24 h at 4°C. Following rinsing, the sections were immersed in 0.05 M Tris-HCl buffer (pH 7.6), containing 0.05% 3,3’ diaminobenzidine (DAB), 0.01% H2O2, and 0.6% nickel ammonium sulfate for 2–5 min at room temperature. The sections were mounted on gelatin-coated glass slides, dried, dehydrated, and covered with a coverslip, using DPX, for light microscopy.
3
Immunohistochemical Detection of c-Fos
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The floating sections were rinsed in 0.01 M PB saline (PBS, pH 7.4), processed 30 min in 0.3% H2O2 in PBS, and incubated in blocking buffer (10% bovine serum in PBS) for 1 h. Then the sections were incubated with rabbit polyclonal antibody against c-Fos (1:6000, sc-253, Santa Cruz Biotechnology, Santa Cruz, USA) in PBS containing 1% bovine serum for 72 h at 4°C on an agitator. After rinsing in PBS, sections were incubated with a biotinylated goat anti-rabbit IgG (1:1000, AP132B, Millipore, Temecula, CA, USA) for 48 h at 4°C and then with horseradish peroxidase conjugated streptavidin (1:2000, SA202, Millipore, Temecula, CA, USA) for 24 h at 4°C. Following rinsing, the sections were immersed in 0.05 M Tris-HCl buffer (pH 7.6), containing 0.05% 3, 3′ diaminobenzidine (DAB), 0.01% H2O2, and 0.6% nickel ammonium sulfate for 2–5 min at room temperature. The sections were mounted on gelatin-coated glass slides, dried, dehydrated and covered with a coverslip, using DPX, for light microscopy.
4
AQP4 ELISA Assay Protocol
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The ELISA was performed as previously described by Pisani et al., 35 . Briefly, Maxisorp NUNC Plates (Thermo) were coated with 0.2 μg of commercial goat anti‐AQP4 antibody (Santa Cruz Biotechnology, sc‐9888) overnight at 4°C or for 2 hrs at 37°C. After coating, wells were washed and coated with approximately 35 ng of AQP4. Negative control wells were only coated with the buffer. After incubation for 1 hr, sera diluted from 1:1000 to 1:8000 and goat anti‐AQP4 were incubated in the wells for 1 hr under shaking. Wells were then washed, incubated with anti‐human biotinylated secondary antibody (Millipore AP112B), washed again and incubated with streptavidin‐HRP (Millipore SA202, 1:1000 in A). After 1 hr, 100 μl of TMB solution was added (Millipore) for 20 min., and the reaction was stopped by adding 100 μl of 0.3M sulphuric acid solution. Finally, absorbance was read at 450 nm. Normalized absorbance was calculated as follows: absorbance of AQP4‐coated well minus absorbance of negative control well. Absorbance was read using a Flex Station 3 (Molecular Devices, Sunnyvale, California, USA).
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