As previously described [43 (link)], brain floating sections were incubated with 10% normal donkey serum for 1 h at room temperature, followed by incubation with appropriate primary antibodies overnight. The following primary antibodies were used in different combinations: anti-NeuN (MAB377, Millipore); anti-Aβ42 and anti-PHF1 (Thermo Fisher); anti-IBA1 (Proteintech Group); anti-P-H2A.X (Cell Signaling); anti-4-HNE and anti-8-OHdG (Abcam Inc.). Sections were then washed four times at room temperature, followed by incubation with proper Alexa Fluor 594/488 donkey anti-mouse/rabbit secondary antibody (Thermo Fisher) for 1 h. After washes, sections were mounted and coverslipped in Vectashield mounting medium with DAPI (Vector Laboratories). All the fluorescence images were captured on an LSM510 Meta confocal laser microscope (Carl Zeiss) using 40×oil immersion Neofluor objectives with the image size set at 1024 x 1024 pixels. The captured images were viewed and analyzed using LSM510 Meta imaging software. At least 4–5 representative sections per animal were utilized for immunostaining and the typical staining was selected for presentation.
Anti iba1
Manufactured by Proteintech
Sourced in United States
Anti-IBA1 is a primary antibody that recognizes the IBA1 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.
Automatically generated - may contain errors
Lab products found in correlation
Anti gfap, by Proteintech (3 mentions)
Lsm510 meta confocal laser microscope, by Zeiss (2 mentions)
Anti 8 ohdg, by Abcam (1 mentions)
Anti ph2ax, by Cell Signaling Technology (1 mentions)
Anti 4 hne, by Abcam (1 mentions)
Anti neun, by Merck Group (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Anti sirt1, by Proteintech (1 mentions)
Nf κb p65 antibody, by Cell Signaling Technology (1 mentions)
Anti tnf α, by Proteintech (1 mentions)
Anti pgc 1α, by Abcam (1 mentions)
Anti il 1β, by Abcam (1 mentions)
Anti bdnf, by Abcam (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Anti cd206, by Proteintech (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Anti ng2, by Merck Group (1 mentions)
Anti synapsin 1, by Cell Signaling Technology (1 mentions)
Anti psd95, by Cell Signaling Technology (1 mentions)
Dab peroxidase substrate, by Beyotime (1 mentions)
11 protocols using anti iba1
1
Immunohistochemical Analysis of Alzheimer's Pathology
2
Neuroinflammation and Cognitive Impairment
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The instruments and reagents used in these experiments were as follows: a 7.0T animal magnetic resonance instrument (Bruker, Germany); novel object recognition and test system (Boster Bioengineering, China); Y labyrinth video analysis system (Shanghai Xinruan Information Technology, China); high-fat feed (21% fat, 0.15% cholesterol, Jiangsu Medisen Biomedicine, China); isoflurane (Shenzhen Reward, China); immunohistochemistry kit and DAB staining kit (Boster Bioengineering, China); and haematoxylin staining solution and eosin staining solution (Beijing Solebao Technology, China). The primary antibodies in this study were as follows: anti-SIRT1 (Proteintech, USA), anti-TNF-α (Proteintech, USA), anti-β-actin (Proteintech, USA), anti-IBA1 (Proteintech, USA), anti-GFAP (Proteintech, USA), anti-PGC-1α (Abcam, USA), anti-BDNF (Abcam, USA), anti-IL-1β (Abcam, USA), NF-κBp65 antibody (Cell Signaling Technology, USA), goat anti-mouse/anti-rabbit secondary antibody (Proteintech, USA), and antibody diluent (Beijing Biyuntian, China).
3
Immunofluorescence Analysis of Spinal Cord Pathology
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Spinal cord paraffin sections (4 μm thick) from each specimen were dewaxed, hydrated, and boiled to repair the antigen. Sections were blocked with 0.1% Triton X-100 and 10% normal goat serum at room temperature for 2 h. Sections were incubated overnight at 4°C with the following primary antibodies: anti-CD16 (1:200; Bioss), anti-CD206 (1:5000; Proteintech), anti-Iba-1 (1:200; Proteintech), and anti-NF-κB P65 (1:200; CST). The sections were rinsed 3 times using PBS, 5 min each time, after which the sections were incubated with secondary antibodies for 1 h at room temperature. Following the PBS rinsing, the nuclei were stained blue with 4′,6-diamidino-2-phenylindole (DAPI) (Solarbio). The sections were sealed with an anti-fluorescence quenching agent after processing, and immunofluorescence imaging was performed using a digital slide scanner. ImageJ software (National Institutes of Health, United States) was utilized to measure the mean fluorescence intensity of specific proteins.
4
Immunohistochemical Analysis of Myelination and Neuroinflammation
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Fixed brains were paraffin-embedded and cut into 4-μm sections. After antigen retrieval, the sections were blocked and stained with anti-Oligo2 (1:500, Proteintech), anti-MBP (1:200, Abcam), anti-NG2 (1:100, Millipore), anti-Iba1 (1:500, Proteintech), anti-GFAP (1:500, Proteintech), anti-MAP2 (1:500, Proteintech), anti-PSD95 (1:500, Cell Signaling Technology), or anti-Synapsin I (1:500, Cell Signaling Technology). Then, the sections were incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (1:500, Abbkine, China) or (HRP)-conjugated anti-mouse antibody (1:500, Abbkine, China). The sections were developed using DAB peroxidase substrate (Beyotime Biotechnology, China). The sections were photographed with an Olympus AH-2 light microscope (× 200; Olympus). Images were imported into ImageJ for quantification. Briefly, the well-stained area on the images was first selected; then, the mean integrated optical density value of the area was calculated by ImageJ. According to the mean integrated optical density of the control group, the relative density of the EAE group and the MCC950 treatment group was calculated.
5
Immunohistochemical Analysis of Rat Brain
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After behavioral testing, the rats were anesthetized with an intraperitoneal injection of pentobarbital sodium. Following cardiac perfusion with 0.9% sodium chloride and 4% paraformaldehyde, the brains were extracted, fixed in 4% paraformaldehyde for 24 h and dehydrated using an alcohol gradient. Coronal paraffin sections with a thickness of 3 μm were obtained from paraffin blocks using a microtome. Subsequent to high-temperature repair and blocking, the sections underwent an overnight incubation at 4°C with the primary antibody, including anti-phospho-TAK1(Thr184/187) (rabbit. # PA5-99340; Thermo Fisher Scientific), anti-Neun (rabbit. # 26978-1-AP; Proteintech), anti-IBA1(mouse. # ab283319; Abcam), anti-glial fibrillary acidic protein (GFAP; mouse. # 60190-1-ig; Proteintech), and anti-GSDMD (rabbit. # 20770-1-AP; Proteintech). After three washes in phosphate buffer solution (PBS), the sections were exposed to appropriate conjugated secondary antibodies: CoraLite488-conjugated Affinipure Goat Anti-Mouse IgG([H+L] SA00013-1; Proteintech) and CoraLite594-conjugated Goat Anti-Rabbit IgG([H+L] #SA00013-4; Proteintech), followed by counterstaining with DAPI for 10 min in the dark. Images were captured using a fluorescence microscope.
6
Immunohistochemical Analysis of IBA1 in Tumor Samples
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Paraffin blocks corresponding to the tumour samples analysed using Raman were processed for immunohistochemistry (IHC). IHC were carried out on the H2P2 core facility. The sections were incubated 1 h at room temperature with anti‐IBA1 (1:10,000 dilution; proteintech). Immunostaining was carried out using the discovery ultra (Ventana Medical Systems) with the Rhodamine kit (a “biotin‐free” system using multimer technology, Roche) and a Tris borate EDTA pH8 buffer for antigen retrieval. Sections were converted on to digital slides with the scanner Nanozoomer 2.0‐RS and immunostaining were quantified with the NIS software (Nikon).
7
Molecular Mechanisms of Neuropathic Pain
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Ipsilateral dorsal lumbar (L4–L6) spinal cord and dorsal root ganglia (DRG) were collected immediately after decapitation on 2nd, 7th, and 14th days after CCI. Tissue was stored at −80°C until processing, which was described previously [28 (link)]. Blots were incubated overnight at 4°C with primary antibodies: anti-IBA-1 (rabbit anti-rat, 1 : 1000, Proteintech, Chicago IL, USA), anti-TLR2 (rabbit anti-rat, 1 : 2000, Novus Biological, Littleton CO, USA), anti-TLR4 (rabbit anti-rat, 1 : 1000, Proteintech, Chicago IL, USA), anti-MyD88 (rabbit anti-rat, 1 : 1000, Abcam, Cambridge, UK), and anti-TRIF (rabbit anti-rat, 1 : 500, Novus Biologicals, Littleton CO, USA) and for 1 h at RT with a corresponding secondary polyclonal HRP antibody (goat anti-rabbit IgG, 1 : 5000, Bio-Rad, Hercules, CA, USA). Both primary and secondary antibodies were diluted in solutions from SignalBoost Immunoreaction Enhancer Kit (Merck Millipore, Darmstadt, Germany). Immunocomplexes were detected using Clarity Western ECL Substrate (BioRad, Hercules, CA, USA) and visualized using a Fujifilm LAS-4000 fluoroimager system. The blots were stripped using Restore Western Blot Stripping Buffer (ThermoScientific, Waltham, MA, USA) for 15 minutes at RT and reprobed with an antibody against GAPDH (mouse anti-rabbit, 1 : 5000, Merck Millipore, Darmstadt, Germany) as a loading control.
8
Microglial Activation Assessment in Spinal Cord
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Immunohistochemistry was performed to assess the degree of microglial aggregation and activation, using ionized calcium-binding adaptor molecule 1 (Iba-1) and CD68, respectively, as markers. After spinal cord sections were deparaffinized, hydrated, and subjected to antigen retrieval, blocking was performed using endogenous peroxidase and serum. Subsequently, the sections were incubated with anti-Iba-1 (rabbit polyclonal antibody against rat, 1:200, ProteinTech Group, Chicago, IL, USA, Cat# 10904-1-AP, RRID: AB_2224377) and anti-CD68 (mouse monoclonal antibody against rat, 1:100, Abcam, Cambridge, UK, Cat# ab31630, RRID: AB_1141557) primary antibodies overnight at 4°C (14–16 hours). Sections were incubated with biotin-labeled anti-mouse/rabbit secondary antibody (Maixin, Fuzhou, Fujian, China, Cat# KIT-9710) at 37°C for 30 minutes the following day, followed by 3,3′-diaminobenzidine staining (Cat# DAB-0031, Maixin). Finally, the sections were observed and images of the immunocomplexes were captured using a Nikon light microscope (Minato-ku, Tokyo, Japan).
9
Immunofluorescence Staining of Brain Sections
10
Immunofluorescence Antibody Staining Protocol
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