Mice were deeply anesthetized by CO2 inhalation and transcardially perfused with sterile PBS followed by 4% formaldehyde. Brains were post-fixed in 4% formaldehyde for 24 h and cryoprotected in 20% sucrose for 48 h. Preserved brains were frozen using dry-ice cooled isopentane and sectioned (25 μm) using a Microm HM550 cryostat. Iba-1 staining was performed as previously described (Wohleb et al., 2011 (link)). In brief, free-floating sections were blocked and then incubated with rabbit anti-mouse Iba-1 antibody (Wako Chemicals) overnight at 4°C. Sections were washed with PBS and incubated with a fluorochrome-conjugated secondary antibody (Alexa Flour 594). Fluorescent images were visualized using an epifluorescent Leica DM5000B microscope and were captured using a Leica DFC300 FX camera and imaging software. Quantitation was assessed using digital image analysis (Donnelly et al., 2009 (link)) in the hippocampus (12 representative images) and prefrontal cortex (6 representative images) at 20× magnification. Threshold staining was determined using NIH ImageJ software. Results are reported as the average percent area for Iba-1+ staining.
Rabbit anti mouse iba 1 antibody
Manufactured by Fujifilm
Sourced in Japan, United States
The Rabbit anti-mouse Iba-1 antibody is a laboratory reagent used to detect the Iba-1 protein, which is a marker for microglia cells in mouse tissue samples. This antibody is produced in rabbits and specifically targets the mouse Iba-1 protein.
Automatically generated - may contain errors
Lab products found in correlation
Dfc300fx, by Leica (1 mentions)
Fv 100d, by Olympus (1 mentions)
Rat anti mouse cd16 32 antibody, by BioLegend (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Neuronal nuclei (neun), by Merck Group (1 mentions)
Confocal laser scanning microscope, by Leica (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
3 protocols using rabbit anti mouse iba 1 antibody
1
Microglia Quantification in Mouse Brain
2
Imaging Neuroinflammation and Neurodegeneration in Ischemic Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were sacrificed 3 days after ischaemia. The brains were removed following transcardial perfusion with 0.9% saline and 4% paraformaldehyde. Brains were postfixed in 4% paraformaldehyde overnight and then cryoprotected by immersion in 20% sucrose for 48 h. Coronal sections (30 μm) were prepared and immunostained as previously described18 (link). In brief, brain slices were incubated with rabbit anti-mouse Iba1 antibody (dilution 1:500; WAKO, Osaka, Japan), mouse anti-mouse NeuN antibody (dilution 1: 500; Millipore, Schwalbach, Germany), and rat anti-mouse CD16/32 antibody (dilution 1:300; Biolegend) overnight at 4 °C. Antibody binding was visualized with Alexa Fluor 488- and Alexa Fluor 594-labelled secondary antibodies (dilution 1:500; Invitrogen, Carlsbad, CA) using a laser confocal microscope (FV-100D; Olympus, Tokyo, Japan). Negative controls were prepared by omitting the primary antibodies and used to adjust confocal machine settings (i.e., gain and black level). The nuclei were stained with DAPI. Cells labelled with NeuN, or double-labelled with Iba1 and CD16/32, were counted in three representative micrographs of each hippocampal region (bregma −2.20 through −2.66) to quantify the fluorescent signal.
3
Quantification of Neuronal and Microglial Populations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After behavioral testing, three mice from each group were anesthetized with
pentobarbital sodium (80 mg/kg) and underwent cardiac perfusion with physiological
saline to obtain clean brain tissue without blood. The dissected tissue was fixed in
4% paraformaldehyde for more than 24 h, frozen, sectioned at 10 μm, and mounted on
slides. After antigen retrieval and blocking of endogenous peroxidase activity,
sections were incubated with monoclonal mouse anti-neuronal nuclei antibody (Neu-N,
1:500, Chemicon, USA) or rabbit-anti-mouse Iba-1 antibody (1:500, Wako Chemicals,
Japan) at 4°C overnight to detect brain neurons and microglia. After that, the
sections were incubated with goat anti-mouse IgG labeled with red-fluorescent Alexa
Fluor 594 (Molecular Probes, USA) or goat-anti-rabbit IgG labeled with
green-fluorescent Alexa Fluor 488 (Molecular Probes) secondary antibodies. At least
10 serial sections from the hippocampus and cortex of three mice were selected,
viewed, and photographed using a laser confocal scanning microscope (Leica, Germany).
The numbers of microglial cells and neurons in the cortex were counted during
microscopic observation, and those in the hippocampal dentate gyrus were calculated
from the fluorescence intensity using Image-Pro Plus 6.0 (Media Cybernetics,
USA).
pentobarbital sodium (80 mg/kg) and underwent cardiac perfusion with physiological
saline to obtain clean brain tissue without blood. The dissected tissue was fixed in
4% paraformaldehyde for more than 24 h, frozen, sectioned at 10 μm, and mounted on
slides. After antigen retrieval and blocking of endogenous peroxidase activity,
sections were incubated with monoclonal mouse anti-neuronal nuclei antibody (Neu-N,
1:500, Chemicon, USA) or rabbit-anti-mouse Iba-1 antibody (1:500, Wako Chemicals,
Japan) at 4°C overnight to detect brain neurons and microglia. After that, the
sections were incubated with goat anti-mouse IgG labeled with red-fluorescent Alexa
Fluor 594 (Molecular Probes, USA) or goat-anti-rabbit IgG labeled with
green-fluorescent Alexa Fluor 488 (Molecular Probes) secondary antibodies. At least
10 serial sections from the hippocampus and cortex of three mice were selected,
viewed, and photographed using a laser confocal scanning microscope (Leica, Germany).
The numbers of microglial cells and neurons in the cortex were counted during
microscopic observation, and those in the hippocampal dentate gyrus were calculated
from the fluorescence intensity using Image-Pro Plus 6.0 (Media Cybernetics,
USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!