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Rabbit anti iba1

Manufactured by Cell Signaling Technology
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Rabbit anti-Iba1 is a primary antibody that specifically recognizes the Iba1 protein, a widely used marker for microglia. Iba1 is a calcium-binding protein expressed in macrophages and microglia and is involved in their activation and motility. The Rabbit anti-Iba1 antibody can be used to detect and study microglia in various experimental settings.

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9 protocols using rabbit anti iba1

1

Immunofluorescent Imaging of Murine Hippocampus

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The procedures were modified from the previous reports (Chen et al., 2021 (link)). Mice were anesthetized with 1% pentobarbital sodium and intracardially perfused with saline followed by 4% polyformaldehyde. The brains were removed, postfixed in 4% polyformaldehyde for 12 h, and then equilibrated in 30% sucrose. Coronal sections of 40-μm thickness containing the hippocampus were collected. Brain slices were blocked for 1 h with 5% bovine serum albumin and 1% Triton X-100 at room temperature and then incubated with primary antibodies (rabbit anti-NeuN 1:300, Cell Signaling Technology, Cat# 12943; mouse anti-GFAP 1:500, Cell Signaling Technology, Cat# 3670 and rabbit anti-Iba1 1:500, Cat# 01919741) overnight at 4°C. The next day, the slices were incubated with secondary antibodies (Alexa 488-conjugated goat anti-rabbit and Alexa 647-conjugated goat anti-mouse, 1:500, Invitrogen) for 2 h at room temperature after washing three times with PBS and then incubated with DAPI (Invitrogen) for 10 min and washed for another three times. The slices were mounted on microscope slides. Fluorescence images were captured using a confocal microscope (A1R, Nikon). Cell counting and fluorescent area calculation were performed by ImageJ.
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2

Western Blotting Analysis of Inflammatory Markers in ICH

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For the western blotting, brain tissue or cultured cells were grinded and homogenized in RIPA (Beyotime) and protease inhibitor (Beyotime). Protein extracted from perilesional tissue was analyzed by western blotting. The penumbra around the hematoma in our study was 0.5 mm thick. Perilesional tissue was extracted randomly from that region. For the control group, brain tissue was harvested from the sham group mice at the same location, where the tissue was dissected from the ICH mice. The same amount of total loading proteins (15–50 g) was separated by SDS/PAGE according to standard protocols and transferred to nitrocellulose membranes. Primary antibodies were used: rabbit anti-Iba-1 (1:1000; Cell Signaling technology), Rabbit anti-TNF-α (1:1000; Abcam), IL-1β (1:500; Abcam), Rat anti-IL-10 (1:1000; Abcam), Rabbit anti-Arginase-1 (1:1000; Cell Signaling technology) for in vivo tissue; Rabbit anti-p-Erk (1:1000; Cell signaling technology), Rabbit anti-p-CREB (1:1000; Cell signaling technology), Rabbit anti-Erk (1:1000; Abcam), rabbit GAPDH (1:2000; Cell Signaling technology). After primary incubation, the membranes were followed incubated within HRP-conjugated anti-rat or anti-rabbit IgG and detected using enhanced ECL detection system (Merck Millipore). Quantification was carried out with ImageJ software (NIH).
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3

Immunofluorescence Staining of Cells

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We captured cells on the cover glass and fixed them with 4% paraformaldehyde (PFA, Invitrogen, CA, USA) for 15 min. The cells were incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies that were fluorescently labelled. The primary antibodies used were as follows: rabbit anti-VE-cadherin (1:250; cat. no. 2500, Cell Signaling Technology, MA, USA), rabbit anti-Iba-1 (1:250; cat. no. ab178846, Abcam, MA, USA), and mouse anti-Thrombospondin 1 (1:250; cat. no. ab1823, Abcam, MA, USA). The secondary antibodies used were Alexa Fluor 488 anti-rabbit IgG (1:500; cat. no. 4412, Cell Signaling Technology, MA, USA) or Alexa Fluor 555 anti-mouse IgG (1:500; cat. no. 4409, Cell Signaling Technology, MA, USA). Afterward, the Nuclei were stained with DAPI for 5 min in the dark. All images were captured using a confocal microscope (Zeiss LSM 980 with Airyscan 2, Oberkochen, Germany) or a fluorescence microscope (Zeiss Axio Imager Z1, Oberkochen, Germany).
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4

Molecular Profiling of Neuroinflammation and Neurodegeneration

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Western Blot was used to confirm findings from TUNEL assay and IHC. Tissue was lysed in radioimmunoprecipitation assay (RIPA) buffer or mitochondria/cytosol fractions were isolated using a Abcam’s Mitochondria/Cytosol Fractionation Kit (Abcam, ab65320, Cambridge, MA) according to the manufacturer’s instructions. Primary antibodies used and their respective dilutions were as follows: rabbit anti-cleaved caspase 3 (c-cas 3, 1:1000) from Cell Signaling Technology (Danvers, MA); rabbit anti-Iba1(1:1000), rabbit anti-GFAP (1:1000); rabbit anti-Aβ (1:1000); rabbit anti-Tau (1:1000), rabbit anti-p-Tau (1:1000), rabbit anti-brain-derived neurotrophic factor (BDNF, 1:1000), rabbit anti-NF-κB (1:1000), rabbit anti-IkBα (1:1000), rabbit anti-histone 3 (H3, 1:1000), mouse anti-β-actin (1:3000) from Abcam.
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5

MPTP-induced Parkinson's Disease Model

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CA (≥98%) was purchased from Yiyan Bio-technology (Shanghai, China); its chemical structure is shown in Figure 1a. MPTP (M0896) was purchased from Sigma (Missouri, MO, USA). The primary antibodies used were mouse anti-TH (MAB318, Millipore, Burlington, MA, USA), rabbit anti- BDNF (ab108319, Abcam, UK), rabbit anti-GFAP (#80788, Cell Signaling, Beverly, MA, USA), rabbit anti-Iba-1 (#17198, Cell Signaling, Beverly, MA, USA), rabbit anti-CD11b (#17800, Cell Signaling, Beverly, MA, USA), mouse anti-β-actin (66009-1-Ig, Proteintech, Chicago, IL, USA) and rabbit anti-GAPDH (10494-1-AP, Proteintech, Chicago, IL, USA). The secondary antibodies used were FITC-conjugated goat anti-mouse IgG (A0568, Beyotime, Shanghai, China) and CY3-conjugated goat anti-rabbit IgG (A0516, Beyotime, Shanghai, China); goat anti-rabbit IgG (15015, Proteintech, Chicago, IL, USA) and goat anti-mouse IgG (15014, Proteintech, Chicago, IL, USA) conjugated to horseradish peroxidase (HRP). The materials for RNA-sequence are indicated in methods below.
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6

Quantifying CNPase, MBP, NF200, and IBA1 in Mouse Brain Regions

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Proteins were extracted from different brain regions (cortex, hippocampus, cerebellum) and spinal cord of WT and IRE1C148S mutant mice as previously described (15 (link)). Equal amounts of each sample (20 g) were loaded on an 8% or 15% SDS-PAGE depending on the size of the protein to be detected. After transfer to nitrocellulose membrane using the TurboBlot system (Biorad), primary antibodies were applied: rabbit anti-CNPase (1:1,000, Cell signaling), rat anti-MBP (1:40.000, Millipore), mouse anti-NF200 (1:1,000, Sigma), rabbit anti-IBA1 (1:500, Cell signaling). Species-specific HRP-conjugated secondary antibodies from ImmunoJax were used. Following incubation with West Pico ECL substrate (Thermo Fisher Scientific), blots were imaged using the Gel Doc XR+ (BioRad) and analyzed with image J software (NIH). Protein quantification was normalized to the total protein for each sample determined after transfer to the membrane using Ponceau S solution (Sigma).
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7

Visualizing Microglial Activation via Iba1 Immunofluorescence

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The protein expression of ionized calcium binding adaptor molecule 1 (Iba1) reflecting the activation of microglia was detected by the immunofluorescence method [36 (link)]. Briefly, fixed brain tissue was dehydrated by sucrose gradient solutions, and the frozen sections were stained after OCT medium embedding. Subsequently, the tissue sections were fixed in paraformaldehyde for 30 min, permeabilized with 0.2% Triton X-100 for 15 min, blocked in 1% bovine serum albumin for 30 min, and incubated with primary antibody rabbit anti-Iba1 (1 : 100, Cell Signaling Technology (CST), Danvers, MA, USA) at 4°C overnight. After washing, the sections were incubated with the corresponding FITC-labeled or Cy3-labeled secondary antibody (Kangwei Century Biotechnology Co., Ltd., Beijing, China) in the dark at 37°C for 60 min. Finally, the tissue sections were incubated for 5 min with DAPI before examining the protein levels under a fluorescence microscope. For quantification, the HE mean number of Iba1-positive cells in hippocampal CA1 area was calculated. Activated microglia were calculated as the percentage of cells with shorter, stubbier processes, namely, activated microglia% = (the number of activated microglia/total number of Iba1‐positive cells) × 100 [36 (link)].
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8

Immunofluorescence Staining of Iba1+ Cells in Mouse Hippocampus

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Immunofluorescence staining was performed following established protocols as described previously [33 (link)]. Briefly, 8-μm-thick coronal sections were fixed in paraformaldehyde for 30 min, permeabilized using 0.2% Triton X-100 for 15 min, blocked with 1% bovine serum albumin for 30 min, and then incubated with the primary antibody rabbit anti-Iba1 (1:100; Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. After rinsing, the sections were incubated with the FITC-labeled goat anti-rabbit secondary antibody (1:200; Affinity, Cincinnati, OH, USA) in a dark setting at 37 °C for 1 h. Finally, the sections were stained with DAPI for 5 min. Three sections from each mouse were observed under a fluorescence microscope (Olympus, Tokyo, Japan). The Iba-1 positive cells/mm2 in the CA1 area of the hippocampus were quantified using Image-Pro Plus v6.0.
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9

Antibody Profiling of Tissue Samples

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The primary antibodies used for western blot are as follows: rabbit anti-HIF-1α (1:1000, Cell Signaling Technology, RRID: AB_2799095), and rabbit anti-LDHA (1:5000, Abcam, RRID: AB_2889291). For western blotting, horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000, Sigma-Aldrich) were used. The primary antibodies used for immunofluorescent staining are as follows: rabbit anti-CD8α (1:200, Abcam, RRID: AB_2890649), rabbit anti-CD4 (1:100, Abcam, RRID: AB_2686917), rabbit anti-GFAP (1:200, Cell Signaling Technology, RRID: AB_2799963), and rabbit anti-Iba1 (1:200, Cell Signaling Technology, RRID: AB_2820254). The secondary antibodies used for immunohistochemical staining were bought from Invitrogen.
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