The brain were fixed in embedding agent (Solarbio) and cut into 5 μm sections. The sections were exposed to the immunized blocking solution (Solarbio) for 1 h. After blocking, the sections were incubated with the primary Iba1 antibody (1:200; Abcam) at 4°C overnight. Then, the sections were washed by PBS and incubated with the fluorescent secondary antibody (CWBIO, Beijing, China) at 25°C for 1 h. Next, the nuclei were stained with DAPI (Thermo Fisher Scientific). Finally, the sections were sealed with anti-quenching seals (Solarbio) and stored at 4°C. The sections were observed under a confocal fluorescence microscope (Nikon).
Iba1 primary antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
Iba1 primary antibody is a protein marker used to detect and identify microglia and macrophage cells in biological samples. It is commonly used in immunohistochemistry and immunofluorescence applications.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Confocal fluorescence microscope, by Nikon (1 mentions)
Fluorescent secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated donkey anti goat igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Application module, by Leica (1 mentions)
Full hd microscopic camera, by Leica (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Cm1950 cryostat, by Leica (1 mentions)
Lsm 880 confocal microscope, by Zeiss (1 mentions)
Nucblue, by Thermo Fisher Scientific (1 mentions)
Abc reagent, by Vector Laboratories (1 mentions)
Antigen unmasking solution, by Vector Laboratories (1 mentions)
Ki 67 primary antibody, by Agilent Technologies (1 mentions)
7 protocols using iba1 primary antibody
1
Immunostaining of Microglial Cells
2
Histological Analysis of Rat Brain
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After the MWM test, the brain of the rat was dissected into two hemispheres, one of which was serially embedded in paraffin and cut into 5-μm thick coronal slices for hematoxylin and eosin (HE) staining, while the other hemisphere was processed with optimal cutting temperature compound (OCT) for immunofluorescence. For HE staining analysis, the brain sections were dewaxed, rehydrated, and stained with hematoxylin and eosin according to the manufacturer’s instructions. For immunofluorescent staining, the sections were labeled by the antibody of ionized calcium-binding adapter molecule 1 (Iba1). Briefly, the sections were blocked by PBS containing 3% donkey serum and 0.3% Triton X-100 for 1 h and then incubated with primary Iba1 antibody (1:500, Abcam) overnight at 4 °C. After that, the sections were rinsed, and the Iba1-labeled sections were incubated with secondary antibody (Alexa Fluor 594®, 1:500, CST) for 1 h at room temperature. Finally, the sections were rinsed with PBS and then counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The staining was monitored under a microscope (IX53, Olympus, Tokyo, Japan). Neuronal cells and Iba-1 labeled microglia were quantified in five coronal sections at 100 μm intervals in each rat.
3
Histological and Immunofluorescent Brain Analysis
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After the MWM test, the brain of the rat was dissected into two hemispheres, one of which was serially embedded in paraffin and cut into 5-μm thick coronal slices for hematoxylin and eosin (HE) staining, while the other hemisphere was processed by optimal cutting temperature compound (OCT) for immunofluorescence. For HE staining analysis, the brain sections were dewaxed, rehydrated, and stained with hematoxylin and eosin according to the manufacturer's instructions. For immunofluorescent staining, the sections were labeled by the antibody of ionized calcium binding adapter molecule 1 (Iba1) or ROS dye dihydroethidium (DHE). Briefly, the sections were blocked by PBS containing 3% donkey serum and 0.3% Triton X-100 for 1 hour, and then incubated with primary Iba1 antibody (1:500, Abcam) overnight at 4°C or DHE for 30 minutes at 37℃. After that, the sections were rinsed, and the Iba1-labeled sections were incubated with secondary antibody (Alexa Fluor 594®, 1:500, CST) for 1 hour at room temperature. Finally, the sections were rinsed with PBS and then counterstained with 4',6-diamidino-2-phenylindole (DAPI). The staining was monitored under a microscope (Olympus, Tokyo, Japan).
4
Microglial Activation Evaluation After GCI
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Inflammatory responses are initiated and worsened by external damage such as cerebral ischemia [33 (link)]. To determine whether carvacrol can reduce hippocampal microglial activation after GCI, we performed staining against Iba1, a specific marker for microglia detection. The general staining protocol is as follows. Brain sections were incubated in Iba1 primary antibody (diluted 1:500, Abcam, Cambridge, UK) containing 0.3% Triton X-100 for 15~18 hours in a 4 °C maintained incubator. Next, they were reacted with Alexa Fluor 488-conjugated donkey, anti-goat, IgG secondary antibody (diluted 1:250; Invitrogen, Grand Island, NY, United States), and counter-stained with DAPI (diluted 1:1000) for two hours. The sections were observed using a fluorescence microscope (Olympus, Shinjuku, Japan).
5
Microglial Activation Analysis in Brain Tissue
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Unstained deparaffinized 5 μm-thick brain tissue sections were cut and processed with 3% hydrogen peroxide for 20 min, washed by PBS, and incubated with ionized calcium-binding adaptor molecule 1 (Iba-1) primary antibody (Abcam, USA, Catalogue No. ab108539) overnight at 4 °C after 1:100 dilution. HRP performed complex and 3,3̀-diaminobenzidine (DAB) were used for detection of Iba+ microglia according to manufacturer’s instructions (Dako, Denmark). After washing by PBS, tissue slides were counter-stained with hematoxylin for microscopic analysis. Micrographs were captured by Full HD microscopic camera processed by Leica application module (Leica Microsystems GmbH, Wetzlar, Germany). The immunohistochemical examination was performed by an experienced investigator who was blinded to samples’ identity to eliminate any bias in the results.
6
Glioma Tissue Analysis in Mice
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28 days post implantation, the glioma‐bearing mice were anesthetized with pentobarbital (50 mg/kg) and perfused with PBS followed by 4% paraformaldehyde (PFA) transcardially. Post‐fixed of the removed brain tissues in 4% PFA for 24 h, dehydrated by a followed sucrose at a concentration of 0.15 M, 0.5 M, and 0.8 M at 4°C. Using a Leica CM1950 cryostat, the brains were embedded in an optimal cutting‐temperature compound (Sakura). Staining of sections was performed according to our previous methods.
26 (link) Tissue sections were incubated with rabbit Ki‐67 primary antibody (CST, CA, 1:100), IBA1 primary antibody (Abcam, CA, 1:100), GFAP primary antibody from mouse(DAKO, CA, 1:100), CD3 primary antibody from rat(Biolegend, CA, dilution 1:100), and rabbit CD68 primary antibody (Biolegend, CA, 1:100) overnight at 4°C. Then, tissue sections were incubated with Alexa 488 or Alexa 555 conjugated anti‐mouse secondary antibodies, or Alexa 647 conjugated anti‐rabbit secondary antibodies for 60 min at room temperature. We examined tissue sections using a Zeiss LSM880 confocal microscope (Zeiss). TableS4 provides the antibodies used for this study.
26 (link) Tissue sections were incubated with rabbit Ki‐67 primary antibody (CST, CA, 1:100), IBA1 primary antibody (Abcam, CA, 1:100), GFAP primary antibody from mouse(DAKO, CA, 1:100), CD3 primary antibody from rat(Biolegend, CA, dilution 1:100), and rabbit CD68 primary antibody (Biolegend, CA, 1:100) overnight at 4°C. Then, tissue sections were incubated with Alexa 488 or Alexa 555 conjugated anti‐mouse secondary antibodies, or Alexa 647 conjugated anti‐rabbit secondary antibodies for 60 min at room temperature. We examined tissue sections using a Zeiss LSM880 confocal microscope (Zeiss). Table
7
Immunohistochemical Analysis of Tumor-Infiltrating Cells
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For immunohistochemistry, the tissue sections from untreated and treated tumour-bearing mice were deparaffinized, rehydrated, and washed. The tissue sections were then heat treated (95 °C) in antigen unmasking solution (Vector Laboratories, Burlingame, CA) for 30 min. Tissue sections were blocked in goat serum (1:10 in PBS) for one hour at room temperature, treated with Ki-67 primary antibody (rat monoclonal, Dako, 1:100) overnight at 4 °C followed by Alexafluor conjugated anti-rat secondary antibody at 1:100 dilution. Nuclei were stained using NucBlue (Invitrogen). Images were captured by the EVOS FL Cell Imaging System fluorescent microscope. For Iba-1, tissue sections were blocked with Iba-1 primary antibody (rabbit monoclonal, abcam, 1:1000) overnight at 4 °C followed by biotinylated anti-rabbit secondary at 1:2000 dilution. Avidin-biotin reaction was complete by incubating the tissues in ABC reagent (Vector Lab) for 30 min followed by DAB substrate reaction. Methyl green was used for staining the nucleus.
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