The rats were killed by intraperitoneal injection (10% chloral hydrated, 1 ml/100 g) and 4% paraformaldehyde (PFA) was used to fix via cardiac perfusion. Coronal brain slices (15 μm thick) were prepared and used for staining. Slices were washed in PBS for 5 min, three times, then blotted in 5% goat serum for 1 h at room temperature. Slices were incubated with the primary antibodies overnight at 4°C. Primary antibodies used: mouse anti-Iba1 (1:200 Sigma American), mouse anti-GFAP (1:200 Sigma American), rabbit anti-iNOS, anti-Arg1, anti-IL-1β, anti-TNF-α, anti-IL-4, anti-IL-6, and anti-IL-10 (1:200; Bioss, Beijing, China). After having been washed in PBS three times, these slices were incubated with secondary antibodies at 37°C for 2 h. Secondary antibodies: Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200; Bioss, Beijing, China), Cy3-conjugated goat ant-mouse IgG (1:200; Bioss, Beijing, China). Then these slices were washed three times again and stained with DAPI (Biyuntian, China) for 5 min. Results were imaged using a 7266-fluorescence microscope (Leica, Japan).
Rabbit anti inos
Manufactured by Bioss Antibodies
Sourced in China
Rabbit anti-iNOS is a primary antibody that binds to the inducible nitric oxide synthase (iNOS) protein. iNOS is an enzyme responsible for the production of nitric oxide (NO) in response to various stimuli. This antibody can be used for the detection and quantification of iNOS in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti arg1, by Bioss Antibodies (3 mentions)
Anti il 4, by Bioss Antibodies (2 mentions)
Anti il 6, by Bioss Antibodies (2 mentions)
Anti il 1β, by Bioss Antibodies (2 mentions)
Anti il 10, by Bioss Antibodies (2 mentions)
Anti tnf α, by Bioss Antibodies (2 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Bioss Antibodies (2 mentions)
7266 fluorescence microscope, by Leica (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Mouse anti iba1, by Bioss Antibodies (1 mentions)
Rabbit anti cd11b, by Abcam (1 mentions)
Rabbit anti cd163, by Bioss Antibodies (1 mentions)
Np 40 lysis buffer, by Beyotime (1 mentions)
Imager 600, by Cytiva (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Sodium dodecyl sulfate polyacrylamide gel electrophoresis, by Merck Group (1 mentions)
Rabbit anti actin, by Santa Cruz Biotechnology (1 mentions)
Amersham imager, by Cytiva (1 mentions)
4 protocols using rabbit anti inos
1
Immunohistochemical Analysis of Neuroinflammation
2
Immunofluorescence Assay for Microglial Markers
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Cells were fixed with 4% paraformaldehyde for 10 min and then washed in phosphate-buffered saline (PBS). After being blocked with 5% bovine serum albumin containing 0.5% Triton X-100 for 1 h at 37°C, the microglia were incubated with the following primary antibodies at 4°C overnight: mouse anti-Iba1 (1:200, Bioss, Beijing, China), rabbit anti-iNOS, anti-Arg1, anti-IL-1β, anti-TNF-α, anti-IL-4, anti-IL-6 and anti-IL-10 (1:200, Bioss). After being washed in PBS three times, the cells were incubated with the following secondary antibodies at 37°C for 1 h: Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200, Bioss) or Alexa Fluor Cy3-conjugated goat anti-mouse IgG (1:200, Bioss). After being washed in PBS three times, the cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI; Biyuntian, China) for 5 min. The cells were then washed in PBS and observed under a 7266-fluorescence microscope (Leica, Japan).
3
Immunohistochemical Analysis of Neuroinflammatory Markers
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The sections were routinely dewaxed to water, incubated with 3% hydrogen peroxide at room temperature for 10 min, placed in citrate buffer at 95°C for 15 min, and cooled to room temperature. The sections were blocked in goat serum for 30 min and incubated overnight at 4°C with the following primary antibodies: rabbit anti‐CX3CL1 (1:500; Bioss), rabbit anti‐CX3CR1 (1:500; Bioss), rabbit anti‐iNOS (1:400; Bioss), rabbit anti‐CD163 (1:500; Bioss), and rabbit anti‐CD11b (1:4000; Abcam). After being washed in PBS solution, the sections were incubated with the secondary antibodies for 1 h. The following secondary antibodies were used: goat anti‐rabbit IgG horseradish peroxidase (HRP) conjugate (ZSGB‐Bio). After being washed in PBS solution, develop color at room temperature with 3,3'‐diaminobenzidine. The nuclei were stained with hematoxylin. The positive area was counted by an unbiased, blinded investigator using ImageJ software.
4
Protein Extraction and Western Blot
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For protein extraction, 1 mL of NP-40 lysis buffer (Biyuntian) was transferred to each well of a six-well plate of microglia on ice for 30 min, and the cells were then centrifuged at 12,000 rpm for 10 min. The soluble protein solutions were then mixed with 5x sample buffer (Biyuntian) and boiled at 90oC for 10 min. Equal amounts of protein (60 μg) from each sample were separated via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Sigma) and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% milk with Tris-buffered saline for 1 h, and then were incubated with the following primary antibodies at 4°C overnight: rabbit anti-iNOS, anti-Arg1 (1:500, Bioss) and rabbit anti-actin (1:1000, Santa Cruz Biotechnology). The membranes were washed three times in Tris-buffered saline with Tween and then incubated with the secondary antibody (peroxidase-conjugated IgG, 1:5000, Santa) at room temperature for 2 h. Binding antibodies were visualized using enhanced chemiluminescence on an imaging system (Amersham Imager 600).
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