Rabbit iba1 antibody

Manufactured by Fujifilm

Sourced in Japan

The Rabbit Iba1 antibody is a primary antibody designed for the detection of the Iba1 (Ionized calcium-binding adapter molecule 1) protein. Iba1 is a calcium-binding protein that is specifically expressed in macrophages and microglia, and is commonly used as a marker for these cell types. The Rabbit Iba1 antibody can be used in various applications, such as immunohistochemistry, immunocytochemistry, and Western blotting, to identify and study Iba1-positive cells.

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Lab products found in correlation

880 inverted confocal airy scan, by Zeiss (2 mentions) Fluor 647, by Thermo Fisher Scientific (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) P1379, by Merck Group (2 mentions) Anti rabbit igg, by Vector Laboratories (1 mentions) 3 3 diaminobenzidine dab peroxidase substrate kit, by Vector Laboratories (1 mentions) Leica dmrb microscope, by Leica Microsystems (1 mentions) Vectastain universal elite kit, by Vector Laboratories (1 mentions) T8787, by Merck Group (1 mentions) Hrp polymer, by Agilent Technologies (1 mentions) Fluoview 1000 confocal microscope, by Olympus (1 mentions) A1 confocal microscope, by Nikon (1 mentions) Normal goat serum (ngs), by Beyotime (1 mentions) Goat anti rabbit antibody, by Beyotime (1 mentions) Paraformaldehyde (pfa), by Beyotime (1 mentions) Cryostat, by Leica camera (1 mentions) Dab kit, by Beyotime (1 mentions)

7 protocols using rabbit iba1 antibody

Microglial Activation Analysis via Iba-1 Immunohistochemistry

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To study microglial activation, we used monoclonal anti-ionized calcium-binding adapter molecule-1 (Iba-1) rabbit antibody (#019–19741, Wako Pure Chemical Industries, Ltd., Japan). Tissue sections were incubated with the primary antibody overnight at 4°C. A biotinylated universal secondary antibody was then applied for 1 h (anti-rabbit IgG, Vector Labs, Inc., Burlingame, CA), followed by an avidin biotin complex according to the manufacturer’s instructions (Vectastain Universal Elite Kit, Vector Labs). We visualized the antibody binding using the 3,3’-diaminobenzidine (DAB) peroxidase substrate kit (Vector Labs). We counterstained with hematoxylin and examined the sections using light microscopy on a Leica DMRB microscope (Leica Microsystems, Bannockburn, IL).

Shu S., Zhang Z., Spicer D., Kulikowicz E., Hu K., Babapoor-Farrokhran S., Kannan S., Koehler R.C, & Robertson C.L. (2019). Administration of a 20-HETE synthesis inhibitor improves outcome in a rat model of pediatric traumatic brain injury. Developmental neuroscience, 41(3-4), 166-176.

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Microglial Morphology Analysis via Immunostaining

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Immunostaining and analyses of microglial morphology were performed on the PFA-perfused slices using ionizing calcium-binding adaptor molecule 1 (Iba1) rabbit antibody (019-19741; Wako). Briefly, brain slices (30 μm thick) were incubated in 10% normal donkey or goat serum in PBST [1 x PBS (11189, Gibco) + 0.5% Tween 20 (P1379, Sigma)] containing 0.3% Triton X 100 (T8787, Sigma) for antigen retrieval and to block any background for 2 h at RT. Slices were incubated with the primary antibody (1:500) at 4°C overnight. After 3 washes with PBST, slices were incubated with the secondary antibody conjugated with Alexa Fluor 488 or Fluor 647 (1:500, Invitrogen) for 2 h at RT. After 5 washes with PBST, the sections were mounted, dehydrated, and cover-slipped. Images were acquired using Zeiss 880 inverted confocal Airy scan.

Zhang Z., Ma Q., Velagapudi R., Barclay W.E., Rodriguiz R.M., Wetsel W.C., Yang T., Shinohara M.L, & Terrando N. (2022). Annexin-A1 Tripeptide Attenuates Surgery-Induced Neuroinflammation and Memory Deficits Through Regulation the NLRP3 Inflammasome. Frontiers in Immunology, 13, 856254.

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Microglial Morphology Analysis via Immunostaining

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Immunostaining and analyses of microglial morphology were performed on the PFA-perfused slices using ionizing calcium-binding adaptor molecule 1 (Iba1) rabbit antibody (019-19741; Wako) . Briefly, brain slices (30 μm thick) were incubated in 10% normal donkey or goat serum in PBST [1 x PBS (11189, Gibco) + 0.5% Tween 20 (P1379, Sigma)] containing 0.3% Triton X 100 (T8787, Sigma) for antigen retrieval and to block any background for 2 hours at RT. Slices were incubated with the primary antibody (1:500) at 4°C overnight. After 3 washes with PBST, slices were incubated with the secondary antibody conjugated with Alexa Fluor 488 or Fluor 647
(1:500, Invitrogen) for 2 hours at RT. After 5 washes with PBST, the sections were mounted, dehydrated, and cover-slipped. Images were acquired using Zeiss 880 inverted confocal Airy scan.

Zhang Z., Ma Q., Velagapudi R., Barclay W.E., Rodriguiz R.M., Wetsel W.C., Shinohara M.L., & Terrando N. (2020). Annexin-A1 tripeptide attenuates surgery-induced neuroinflammation and memory deficits through regulation of the NLRP3 inflammasome.

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Immunohistochemical Analysis of Rat Cerebellum

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Rats at P7, P9, P11, P15 and P30 were anesthetized following animal welfare committee guidelines, transcardially perfused with PBS followed by 4% paraformaldehyde (PFA). Cerebellums were dissected, fixed with 4% PFA overnight at 4°C, paraffin embedded and processed for histological staining. Sections were cut at 5–10 μm and stored at room temperature. For immunohistochemistry, sections were proceeded as previously described (Endesfelder et al., 2013 (link)). Polyclonal mouse antibody to NG2 chondroitin sulfate proteoglycan (Chemicon, Temecula CA) was diluted 1:500. Monoclonal mouse antibody to myelin basic protein (MBP) (Covance, VA) was diluted 1:500. The monoclonal rabbit Ki67 antibody (Leica Biosystems, Newcastle, UK) was diluted 1:200. Monoclonal mouse CC1 antibody was diluted 1:500 (Abcam, Cambridge MA; Calbiochem, Darmstadt, Germany). Rabbit Iba1 antibody (Wako Chemicals, Richmond VA) was diluted 1:750. All secondary antibodies used were from Jackson Immunoresearch Laboratories, West Grove PA, in carrier solution: FITC-conjugated goat anti-mouse IgG (1:200), FITC-conjugated goat anti-rabbit IgG (1:200), CY3/Rhodamine conjugated goat anti-mouse IgG (1:200) and CY3/Rhodamine conjugated goat anti-rabbit IgG (1:200). Stained sections were mounted in Vectashield mounting medium containing DAPI.

Scheuer T., Brockmöller V., Knowlton M.B., Weitkamp J.H., Ruhwedel T., Mueller S., Endesfelder S., Bührer C, & Schmitz T. (2015). Oligodendroglial maldevelopment in the cerebellum after postnatal hyperoxia and its prevention by minocycline. Glia, 63(10), 1825-1839.

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Quantifying Hepatocellular Lesions in Mice

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Anti-IBA1 immuno-stains were performed on 4 μm sections. After de-waxing and re-hydration in ethanol, antigen de-masking was done in sodium citrate buffer in a water bath at 95°C for 45 minutes. Endogenous peroxidases were quenched with a 5 min treatment in 3% H₂O₂. Slides were incubated with rabbit IBA1 antibody (Wako, 019–19741) diluted 1:500, and developed with HRP polymer (DAKO). Slides were finally counterstained with hematoxylin and mounted with Eukitt. The histological classification of hepatocellular proliferative lesions was performed according to Thoolen et al. [33 (link)]. For each mouse, either the composition of the tumor (in terms of percentage of adenoma and/or carcinoma) or the number of hepatocellular adenomas, early carcinomas (defined as adenomas containing focus of arising carcinoma), carcinomas, and the total number of neoplastic lesions were evaluated by a mouse pathologist. Samples were coded without reference to experimental group and examined blindly.

Collino A., Termanini A., Nicoli P., Diaferia G., Polletti S., Recordati C., Castiglioni V., Caruso D., Mitro N., Natoli G, & Ghisletti S. (2018). Sustained activation of detoxification pathways promotes liver carcinogenesis in response to chronic bile acid-mediated damage. PLoS Genetics, 14(5), e1007380.

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Immunofluorescent Labeling of Brain Tissue

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Eight to ten week old animals were cardiac perfused with phosphate buffered saline (PBS) and paraformaldehyde (PFA). The brain was extracted, post-fixed in PFA for 1 h at 4°C, placed overnight in 30% sucrose and frozen at -80°C. Coronal sections (40 μm) were cut, washed with PBS, and permeabilized with 0.1% Triton X-100, 5% horse or goat serum in PBS. Unless otherwise indicated the antibodies used were from Millipore, Temecula, CA, USA. We used mouse NeuN antibody, 1:1000, rabbit NG2 antibody, 1:1000, rabbit GFAP antibody (Sigma, St. Louis, MO, USA), 1:1000, and rabbit Iba1 antibody (Wako Chemicals, Richmond, VA, USA), 1:500. Secondary antibodies conjugated to either Alexa 633 or 568 were used. Sections were mounted and visualized on either Fluoview 1000 confocal microscope (Olympus, Center Valley, PA, USA) or A1 confocal microscope (Nikon, Tokyo, Japan). Venus fluorescence was detected at 515 nm.

Foley J., Blutstein T., Lee S., Erneux C., Halassa M.M, & Haydon P. (2017). Astrocytic IP3/Ca2+ Signaling Modulates Theta Rhythm and REM Sleep. Frontiers in Neural Circuits, 11, 3.

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Microglial Activation Assessment in Mouse Brain

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On postoperative days 3, 7, and 14, mice were anesthetized with 4 % chloral hydrate and 0.1 % lidocaine and transcardially perfused with 0.9 % ice-cold saline followed by 2.5 % paraformaldehyde (PFA, Beyotime Institute of Biotechnology, Shanghai, China). Brains were harvested and postfixed in 2.5 % PFA. The samples were then cryoprotected in 30 % sucrose solution and embedded in optimal cutting temperature compound (OCT, Xingzhi Biological technology co., LTD, Guangzhou, China). Coronal sections (25 μm) were obtained with a cryostat (Leica, Germany). Sections were blocked in 10 % normal goat serum (Beyotime Institute of Biotechnology) for 30 min at room temperature and incubated with a rabbit Iba1 antibody (1:500, Wako, Japan) at 37 °C for 1.5 h and then at 4 °C overnight. After PBS wash, sections were incubated in goat anti-rabbit antibody (1:300, Beyotime Institute of Biotechnology, China) for 1 h at 37 °C and then in horseradish peroxidase streptavidin (1:200) and visualized with DAB kit (both from Beyotime Institute of Biotechnology). Images were obtained with a microscope (Leica, Wetzlar, Germany). Iba1-positive cells were analyzed using Image-Pro-Plus® 6.0 Software; the cell body to cell size ratio was used to assess microglial activation [25 (link)].

Li Y., Pan K., Chen L., Ning J.L., Li X., Yang T., Terrando N., Gu J, & Tao G. (2016). Deferoxamine regulates neuroinflammation and iron homeostasis in a mouse model of postoperative cognitive dysfunction. Journal of Neuroinflammation, 13, 268.

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