Rats were deeply anesthetized with pentobarbital and transcardially perfused with 4% paraformaldehyde (PFA) at desired time points post-eFSE. Brains were removed and post-fixed in 4% PFA for 90 min. Brains were then cryoprotected in 30% sucrose, rapidly frozen, and stored at −80°C. Thirty micrometer sections of dorsal hippocampus were obtained on a cryostat and stored in antifreeze at 4°C until use. Serial sections were blocked in 10% normal goat serum and 0.03% Triton X in 1× PBS for 1 h at 4°C. Primary antibodies were incubated in 4% normal goat serum with 0.03% Triton X overnight at 4°C. The following antibodies were used: rabbit anti-HMGB1 1:1000 (Abcam), mouse anti-GFAP 1:3000 (Millipore), and mouse anti-IBA1 1:4000 (Wako). Sections were washed with 1× PBS, and the reaction product was visualized using 3,3'-diaminobezidine. Colocalization of cell markers with HMGB1 was achieved by coincubating rabbit anti-HMGB1 1:1000 (Abcam) with the following antibodies: mouse anti-NeuN (Chemicon), mouse anti-GFAP 1:3000 (Millipore), and mouse anti-CD11b (ABD Serotec). After 24 h of incubation, sections were washed in 1× PBS and then incubated in the appropriate secondary antibodies conjugated with Alexa Fluor 568 or Alexa Fluor 488. Colocalization was visualized using confocal microscopy.
Mouse anti cd11b
Manufactured by Bio-Rad
Sourced in United Kingdom
Mouse anti-CD11b is a primary antibody that recognizes the CD11b antigen, also known as the integrin alpha M chain or complement receptor 3 (CR3) alpha subunit. CD11b is expressed on the surface of various immune cells, including monocytes, macrophages, granulocytes, and natural killer cells. This antibody can be used for the identification and enumeration of CD11b-positive cells in applications such as flow cytometry and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti neun, by Merck Group (3 mentions)
Mouse anti gfap, by Merck Group (2 mentions)
Biotinylated secondary antibody, by Vector Laboratories (2 mentions)
Anti mouse cd68, by Bio-Rad (2 mentions)
Mouse anti iba 1, by Fujifilm (1 mentions)
Rabbit anti hmgb1, by Abcam (1 mentions)
Oct medium, by Thermo Fisher Scientific (1 mentions)
Anti calbindin, by Merck Group (1 mentions)
Fluorescence microscope, by Leica camera (1 mentions)
Alexa fluor 594, by Jackson ImmunoResearch (1 mentions)
3 3 diaminobenzidine tetrahydrochloride, by Vector Laboratories (1 mentions)
Mouse anti th, by Merck Group (1 mentions)
Abc elite kit, by Vector Laboratories (1 mentions)
Avidin biotin complex, by Vector Laboratories (1 mentions)
Mouse anti gfap, by Bio-Rad (1 mentions)
Leica fluorescence microscope, by Leica camera (1 mentions)
Goat serum, by Boster Bio (1 mentions)
Goat anti mouse alexa fluor 594, by Abcam (1 mentions)
Goat anti rabbit alexa fluor 555, by Abcam (1 mentions)
Cm3050, by Leica Microsystems (1 mentions)
8 protocols using mouse anti cd11b
1
Immunohistochemical Visualization of HMGB1 in Rat Hippocampus
2
Immunohistochemical Visualization of Brain Cells
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Detections of glial cells and calbindin-positive neurons were carried out in the brain stem and cerebellum tissues by immunohistochemistry. In detail, anesthetized rats were transcardially perfused with saline followed by 4% paraformaldehyde. The rat brains (containing the cerebrum, cerebellum, and brain stem) were dissected and fixed in 4% paraformaldehyde for 6 h, and then immersed in 30% sucrose in PBS for 48 h, flash-frozen, and mounted in OCT medium (Thermo, United States). Subsequently, 15 μm-thick sections of the brain stem and cerebellum were cut and mounted on glass slides. After 1 h of blocking (10% normal goat serum containing 0.3% Triton X-100 in 0.01 M PBS), the sections were immunostained with mouse anti-CD11b (for microglia, 1:200; Serotec, United Kingdom), anti-GFAP (for astrocytes, 1:400; Serotec, United Kingdom) or anti-calbindin (1:2000; Sigma-Aldrich, United States) for 48 h at 4°C. After rinsing in 0.01 M PBS, the sections were incubated overnight at 4°C in goat secondary antibodies conjugated to FITC (1:200, Jackson, United States) or Alexa Fluor®594 (1:400, Jackson, United States), respectively. Finally, images were observed under a fluorescence microscope (Leica, Germany).
3
Immunohistochemical Analysis of Cellular Markers
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Free-oating sections were pretreated with 0.3% H 2 O 2 in 0.1 M PBS (pH 7.2-7.5) for 10 min at RT (60 rpm) to block endogenous peroxidase activity, and washed with 0.1 M PBS for 3 times. The tissue was then blocked with diluted blocking serum (Elite ABC kit, Vector Laboratories, Burlingame, CA, USA) for 20 min at room temperature. Sections were then incubated with the primary antibody against TH (mouse anti-TH, 1:4000, Sigma) or CD11b (mouse anti-CD11b, 1:1000, Serotec) overnight at 4°C. Then the sections were washed and incubated with diluted biotinylated secondary antibody (Vector laboratories) for 30 min, followed by avidinbiotin complex (Vector laboratories) for 30 min at room temperature. Finally, the sections were developed with 3,3'-diaminobenzidine tetra-hydrochloride (Vector Laboratories), mounted onto glass slides for drying overnight, and dehydrated in a gradient of ethyl alcohol and xylene the next day. The slides were sealed with permount mounting medium.
4
Immunohistochemical Analysis of BNIP3L
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After retrieval of brain tissue, specimens were fixed in 4% PFA for 24 h and dehydrated
in 15% and 30% sucrose in turn. Frozen coronal sections of 15 µm thickness embedded in
optimal cutting temperature compound (OCT) were prepared for staining. Air-dried slices
were washed with 0.3% Triton X-100 three times and blocked with 10% goat serum for 2 h at
room temperature for the purpose of reducing nonspecific staining. These slices were
incubated overnight at 4°C with the primary antibody anti-BNIP3 L (1:100, Abcam,
Cambridge, UK) and the following different markers: mouse anti-NeuN (1:400, Millipore,
Bedford, MA, USA), mouse anti-GFAP (1:200, Bio-Rad, Oxford, UK) and mouse anti-CD11b
(1:200, Bio-Rad). After incubation with secondary antibodies, 4,6-diamidino-2-phenylindole
dihydrochloride (DAPI) was used for staining the cell nuclei. Finally, the sections were
observed with a Leica fluorescence microscope (Leica, Wetzlar, Germany) at 200×
magnification and photographed by LAS X software.
in 15% and 30% sucrose in turn. Frozen coronal sections of 15 µm thickness embedded in
optimal cutting temperature compound (OCT) were prepared for staining. Air-dried slices
were washed with 0.3% Triton X-100 three times and blocked with 10% goat serum for 2 h at
room temperature for the purpose of reducing nonspecific staining. These slices were
incubated overnight at 4°C with the primary antibody anti-BNIP3 L (1:100, Abcam,
Cambridge, UK) and the following different markers: mouse anti-NeuN (1:400, Millipore,
Bedford, MA, USA), mouse anti-GFAP (1:200, Bio-Rad, Oxford, UK) and mouse anti-CD11b
(1:200, Bio-Rad). After incubation with secondary antibodies, 4,6-diamidino-2-phenylindole
dihydrochloride (DAPI) was used for staining the cell nuclei. Finally, the sections were
observed with a Leica fluorescence microscope (Leica, Wetzlar, Germany) at 200×
magnification and photographed by LAS X software.
5
Quantification of Microglia in Ischemic Brain
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Rats were perfused with 4% paraformaldehyde. Frozen coronal slices (14 μm) were obtained with a cryostat (CM3050S; Leica Microsystems, Wetzlar, Germany). Tissue sections were incubated with 0.3% Triton X-100 (Sigma-Aldrich) and 5% goat serum (Boster, China) for 1 h at 37°C. The sections were incubated with the following primary antibodies at 4°C overnight: rabbit anti-Iba-1 (1 : 1000, Wako, Japan) and mouse anti-CD11b (1 : 200, Bio-Rad, Hercules, CA). Then, the sections were washed with PBS and incubated for 1 h at room temperature with fluorescent conjugated secondary antibodies, goat anti rabbit Alexa Fluor 555 (1 : 800, Abcam, Cambridge), goat anti-mouse Alexa Fluor 594 (1 : 800, Abcam, Cambridge). The nuclei were stained with DAPI (1 : 5000, Sigma-Aldrich) for 5 min at room temperature. The tissue sections were viewed with a confocal laser scanning microscope (Eclipse-Ti, Nikon, Japan). Counts are expressed as the number/mm2 in images at 40× magnification. Five fields of cortical microglia in the ischemic penumbra were scanned. A researcher blinded to the treatment groups used imaging analysis software (NIS-Elements Viewer 4.50) to count the positive cells.
6
Immunofluorescent Characterization of Ang-2 and MMP-9 Expressions
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To identify where Ang-2 and MMP-9-positive signals were expressed, ANG-2 and MMP-9 was co-stained with NeuN (neuronal marker), glial fibrillary acidic protein (GFAP, astrocyte marker), CD11b (microglial marker), or Lectin (blood vessel marker). In addition, a single immunofluorescent staining for VEGF was conducted to observe its distribution in the hippocampus. Three sections per animal were washed in PBS with 0.15% Triton X-100 and then incubated in blocking serum, 5% normal horse serum in 0.15% triton with PBS. The sections were then incubated in primary antibody cocktail for 22 hours at room temperature. For primary antibody, mouse anti-NeuN (1:1000, Millipore), mouse anti-GFAP (1:500, BD bioscience, San Jose, CA), mouse anti-CD11b (1:500, Bio-Rad, Hercules, CA), rabbit anti-MMP-9 (1:100, Millipore), goat anti-Ang-2 (1:200, Santa Cruz), and FITC-conjugated Tomato-Lectin antibody (1:500, Vector, Burlingame, CA), mouse anti-VEGF antibody (1:500, Santa Cruz) were used. Sections were washed in PBS with 0.15% Triton X-100 and incubated in the Alexa® fluore conjugated secondary antibody cocktail solution (1:200, Invitrogen, Waltham, MA) for 2 hours at room temperature. Stained sections were mounted on resin-coated slides and dried for 30 min. Anti-fade solution (Prolong gold, Invitrogen) was used to maintain the fluorescence.
7
Immunohistochemistry of Brain Injury in Mice
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Immunohistochemistry was performed on 20 µm thick coronal sections from perfused mouse brains. The sections were incubated overnight at 4 °C with primary monoclonal antibody anti-mouse GFAP (0.5 µg/ml, 1:2000; Millipore, Billerica, MA, USA), anti-mouse CD11b (at 1w and 5w, 1.25 µg/ml, 1:800; Bio rad, Hercules, CA, USA) or anti-mouse CD68 (1.0 mg/ml, 1:200; Bio rad, Hercules, CA, USA). Biotinylated secondary antibodies (7.5 µg/ml, Vector Laboratories, Burlingame, CA, USA) were used. GFAP, CD11b and CD68 immunopositive cells were identified by reaction with 3,3 diaminobenzidine-tetrahydrochloride (DAB, Vector Laboratories, Burlingame, CA, USA) as previously described41 (link). Negative control studies, without the primary antibody, were performed in parallel.
The ipsilateral cortex was analyzed over an area included within a 350 µm radius from the contusion edge. Images were analyzed using Fiji software by segmenting the positive signal. GFAP, CD11b and CD68 immunostained area were expressed as positive pixels/total assessed pixels and reported as the percentage of total stained area18 (link).
The ipsilateral cortex was analyzed over an area included within a 350 µm radius from the contusion edge. Images were analyzed using Fiji software by segmenting the positive signal. GFAP, CD11b and CD68 immunostained area were expressed as positive pixels/total assessed pixels and reported as the percentage of total stained area18 (link).
8
Immunofluorescent Staining of Mouse Brain
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Brains were collected after transcardiac perfusion with 30 mL PBS 0.1 M and 60 mL PAF 4% and frozen in isopenthane, 3 min at − 45 °C. Frozen brains were serially cut at the cryostate into 20 μm coronal sections. Before immunofluorescence, tissues were post-fixated with acetone followed by ethanol 100%, 20 s each. Sections were then washed three times with PBS 0.01 M. Immunofluorescence was performed according to the previously described method [15 ]. Primary antibodies used were anti-mouse CD11b (1:30000, BioRad) and anti-mouse CD68 (1:200, Serotec, Kidlington, UK). Secondary antibodies used were Alexa 546 anti-rat (1:500, Invitrogen, Carlsbad, CA) and biotinylated anti rat (1:200, Vector Laboratories, Burlingame, CA), this latter followed by fluorescent signal coupling with streptavidine TSA amplification kit (cyanine 5, Perkin Elmer, MA, USA). Appropriate negative controls were run without the primary antibodies. None of the immunofluorescence reactions gave unspecific fluorescent signal in the negative controls.
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