Brain sections or cultured astrocytes on coverslips were blocked with 3% bovine serum albumin (BSA) in PBS containing 0.3% Triton X-100 for 1 h at room temperature (RT). The sections or cells were then incubated with primary antibodies overnight at 4°C. Two antibodies were added simultaneously for double-immunofluorescence staining. The following antibodies were used: rabbit anti-S100a10 (1:100; Novus, Colorado Springs, CO, United States), mouse anti-C3 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, United States), rabbit anti-Iba-1 (1:800; Sigma, United States), mouse anti-GFAP (1:500; Abcam, Cambridge, United Kingdom), and rabbit anti-GFAP (1:500; Abcam). The samples were then incubated with species-specific secondary antibodies conjugated with Alexa Fluor (1:200; Zhuangzhi, Beijing, China) for 2 h at RT, and nuclei were stained with Hoechst-33342 (1:10000, GeneCopoeia, United States). Fluorescent signals were visualized under a confocal laser microscope.
Rabbit anti iba1
Manufactured by Merck Group
Sourced in United States
Rabbit anti-Iba1 is a polyclonal antibody that specifically binds to the Iba1 protein, which is a calcium-binding adaptor molecule that is predominantly expressed in microglia cells in the central nervous system. The antibody can be used to detect and visualize Iba1-positive cells in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti c3, by Santa Cruz Biotechnology (1 mentions)
Hoechst 33342, by GeneCopoeia (1 mentions)
Mouse anti gfap, by Abcam (1 mentions)
Rabbit anti gfap, by Abcam (1 mentions)
Mouse anti gfap, by Agilent Technologies (1 mentions)
Alexa fluor 488 conjugated goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Ds qi2 camera, by Nikon (1 mentions)
Ts2 s sm microscope, by Nikon (1 mentions)
Mouse antiluciferase, by Merck Group (1 mentions)
Mouse anti parvalbumin, by Merck Group (1 mentions)
Alexa fluor 647 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Mouse anti cre, by Merck Group (1 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
Lsm 510 meta confocal microscope, by Zeiss (1 mentions)
Mouse anti map2, by Merck Group (1 mentions)
Rabbit anti neun, by Merck Group (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (1 mentions)
8 protocols using rabbit anti iba1
1
Astrocyte and Microglia Immunostaining Protocol
2
Quantifying Gliosis Dynamics in Brain Tissues
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To confirm changes in gliosis, immunofluorescence analysis was performed as previously described [32 (link)]. After washing in PBS-T (0.4% Triton X-100 in PBS), brain sections were blocked in blocking solution (1% BSA and 3% normal goat serum in 0.4% PBS-T) at room temperature for 1 h. Then, slices were incubated overnight at 4 °C in PBS-T solution with the following primary antibodies: mouse anti-GFAP (1:500, DAKO, Centennial, CO, USA) and rabbit anti-Iba1 (1:500, Sigma, St. Louis, MO, USA). After washing in PBS-T, the tissues were incubated with Alexa Fluor 594-conjugated goat anti-rabbit (1:500, Invitrogen, Carlsbad, CA, USA) or Alexa Fluor 488-conjugated goat anti-mouse (1:500, Invitrogen) antibodies for 1 h at room temperature, with DAPI as a counterstain.
All images were captured using a Nikon TS2-S-SM microscope (Nikon Microscopy, Tokyo, Japan) equipped with a Nikon DS-Qi2 camera and further analyzed using ImageJ 1.50 software (National Institutes of Health, Bethesda, MD, USA). The penumbra area (including striatum and cortex) was selected as a region of interest for IHC, the intensity of the fluorescence signal was measured and converted into a percentage (n = 3–4 mice per group).
All images were captured using a Nikon TS2-S-SM microscope (Nikon Microscopy, Tokyo, Japan) equipped with a Nikon DS-Qi2 camera and further analyzed using ImageJ 1.50 software (National Institutes of Health, Bethesda, MD, USA). The penumbra area (including striatum and cortex) was selected as a region of interest for IHC, the intensity of the fluorescence signal was measured and converted into a percentage (n = 3–4 mice per group).
3
Immunohistochemical Staining of Brain Sections
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Brain sections were post-fixed with 4% PFA for 10 minutes and then washed with PBS three times and incubated 30 minutes with blocking solution (PBS/0.1% Triton X-100 containing 10% normal goat serum (Sigma-Aldrich)) and then incubation overnight at 4°C in blocking solution with primary antibodies: mouse anti-CRE (Sigma, F3165–2MG, 1:1000); mouse anti-Luciferase (Sigma, F3165–2MG, 1:1000); mouse anti-Parvalbumin (Sigma, F3165–2MG, 1:1000); mouse anti-MAP2 (Sigma, F3165–2MG, 1:1000); rabbit anti-NeuN (Sigma, F3165–2MG, 1:1000); rabbit anti-IBA1 (Sigma, F3165–2MG, 1:1000). Sections were washed with PBS and incubated for 2h at RT with the secondary antibodies: goat anti-mouse IgG Alexa Fluor 488 (Thermo Fisher, A31560, 1:500) and goat anti-rabbit IgG Alexa Fluor 647 (Invitrogen, A32728, 1:1000) diluted in blocking solution. The sections were washed with PBS and incubated during 10 minutes with DAPI (1:5,000; Sigma), washed, and mounted with Vectashield Antifade Mounting Medium (Vector Labs, H-1000). Immunofluorescence was visualized and imaged with a Keyence BZ-X810 microscope, a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss MicroImaging), equipped with EC Plan-Neofluar 40x/1.30 Oil DIC M27 (420462–9900) and Plan-Apochromat 63x/1.40 Oil DIC M27 (420782–9900) objectives and LSM Image software.
4
Immunohistochemical Staining of Brain Tissue
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Free-floating brain sections were washed in phosphate-buffered saline (PBS; Gibco Invitrogen, Waltham, MA, USA) for 20 min. Sections were then incubated in target retrieval solution (Dako, Glostrup, Denmark) at 85°C for 40 min. Afterward, sections were washed for 20 min in PBS, followed by blocking of endogenous peroxidases (3% H2O2 in PBS, Sigma Aldrich) for 10 min and then washed with PBS containing 0.02% Triton-X-100 (PBS-T; Sigma Aldrich). Next, sections were incubated with polyclonal primary rabbit anti-GFAP (1:500; Dako), rabbit anti-Iba1 (1:1,000; Sigma), rabbit anti-CD31 (1:500, Abcam), or polyclonal rabbit anti-AQP4 (1:500; Boster Biological Technology, Ref#PB9475) overnight at 4°C. The next day, sections were washed in PBS-T and subsequently incubated in polyclonal secondary biotinylated goat-anti-rabbit antibody (1:250; Vector Laboratories, Olean, NY, USA) in PBS-T for 2 h at room temperature. Sections were washed in PBS-T followed by incubation in ABC elite solution (1.5% solution A + 1.5% solution B in PBS, Vector Laboratories) for 1 h at room temperature. Afterward, sections were washed in PBS-T for 20 min, and immunolabeling was performed by using 3,3-diaminobenzidine solution (Acros Organics, Geel, Belgium). Sections were washed in distilled H2O and PBS; mounted on gelatin-coated slides; dehydrated in 95%, 99% alcohol, xylene; and coverslipped.
5
Retinal Vasculature Morphology Analysis
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Mice were killed by CO2 inhalation, and the eyes were enucleated and fixed in 4% paraformaldehyde for 30 min at room temperature. After several washes in PBS, the cornea and lens were removed and the retina was carefully detached from RPE/choroid/sclera. The retinas were incubated overnight with primary antibodies (goat anti-collagen IV, 1:100, Serotec, USA; rabbit anti-Iba1, 1:400, 1:500, Millipore, USA) in PBS supplemented with 0.5% Triton X-100, followed by incubation with appropriate Alexa-coupled secondary antibodies (Life Technologies). The retinas were flatmounted and viewed with a fluorescence microscope (DM5500B, Leica, France) or with a confocal microscope (FV1000, Olympus, France).
All cell counting was performed in the inner retina (GCL/IPL). Vessel lengths and vascular areas were calculated with the “angiogenesis tube formation” add-in [14 (link)] available on MetaMorph 2D software (Molecular Devices, France).
All cell counting was performed in the inner retina (GCL/IPL). Vessel lengths and vascular areas were calculated with the “angiogenesis tube formation” add-in [14 (link)] available on MetaMorph 2D software (Molecular Devices, France).
6
Immunohistochemical Profiling of Mouse Brain
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Adult mice were anesthetized and transcardially perfused with 2% paraformaldehyde. Brains were dissected and post fixed for 2–4 h, cryoprotected in 20% sucrose and stained using standard protocols. The following primary antibodies were used: rabbit anti-NG2 (1:200; Millipore), rabbit anti-Olig2 (1:200; Millipore), mouse anti-Olig1 (1/100, R and D Systems), mouse anti-CC1 (1:100; Abcam), mouse anti-VGluT1 (1:500; Millipore), rabbit anti-Iba1 (1:100; Millipore), rat anti-PDGFRα (1:200; Santa Cruz) and rabbit anti-MBP (1:100; Millipore). Edu (Invitrogen), a BrdU analog, was injected intraperitoneally (75 mg/kg) every 2 h for 10 h before sacrifice at 4 dpi. Edu staining was detected with the Click-It™ Kit (Invitrogen). For electron microscopy analysis, Wt brains were processed as previously described (Tepavcevic et al., 2011 (link)) and imaged using a Siemens electron microscope.
7
Spinal Cord Immunohistochemistry of Nicotinic Receptors
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Spinal cords from the rats (3 groups, n = 4) were harvested after perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer (PBS, pH 7.4), dehydrated with 30% sucrose, placed in tissue-embedding gel, and cut into 20-μm spinal cord slices by cryosection. Sections were incubated with primary antibodies (rat anti-nAChR α7 and rabbit anti-Iba1; Millipore, USA) overnight and, after 3 washes (PBS), were incubated with fluorescent-conjugated secondary antibodies (anti-rat-rhodamine and anti-rabbit-FITC; Jackson ImmunoResearch Lab, USA) for one hour. Double-stained dorsal horn sections of the spinal cord were imaged under a Leica fluorescence microscope. nAChR α7-positive neurons and Iba1-stained microglia were counted in a fixed-size area (1600 × 1200 pixels) in the dorsal horn of the spinal cord.
8
Quantifying Cellular Markers in Rat Striatum
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Western blotting was carried out for the marker proteins for each cell type examined. Rats were killed by decapitation after being anesthetized, and the striatum was extracted and homogenized in a lysis buffer to which protease inhibitors had been freshly added. The homogenate was centrifuged at 1500 g for 25 min, and protein concentration was determined by using a Bio-Rad DC protein assay (Bio-Rad, Hercules, CA). Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1% SDS-PAGE) and transferred to PVDF membranes (Millipore). Membranes were incubated in blocking buffer (5% skim milk in TBST), then with mouse anti- NeuN (1∶2000, Millipore), rabbit anti- Darpp32 (1∶1000, Millipore), mouse anti-GFAP (1∶2000, Santa Cruz Biotechnology), and rabbit anti- Iba-1 (1∶2000, Millipore), or rabbit anti -β-actin (1∶2000, Millipore) in TBST overnight at 48°C. Incubated membranes were then treated with secondary antibody conjugated with horseradish peroxidase in TBST for 2 hr at 37°C. Blots were developed by enhanced chemiluminescence and digitally scanned. The optical density of each resulting labeled band was measured in an image analysis program (Image pro-Plus 6.0).
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