Aortic sections were cryosectioned in 10‐μm intervals. After antigen retrieval and blocking with normal goat serum, the slices were incubated with primary antibodies against CD16/32 (ab25235; Abcam) and inducible nitric oxide synthase (ab178945; Abcam) overnight at 4° and then with appropriate fluorescence‐conjugated secondary IgG antibodies (Beyotime Biotechnology) for 60 minutes in the dark at room temperature. 4',6‐Diamidino‐2‐phenylindole was used to stain the cell nucleus. Fluorescence images were taken using a fluorescence microscope (BX53; Olympus). The CD16/32‐positive area and inducible nitric oxide synthase–positive area were measured using ImagePro Plus.
Ab25235
Manufactured by Abcam
Sourced in United States
Ab25235 is a laboratory equipment product designed for specific research applications. It serves a core function without further interpretation of its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Ab5076, by Abcam (2 mentions)
Ab178945, by Abcam (1 mentions)
Ab97779, by Abcam (1 mentions)
Rat anti cd31, by BD (1 mentions)
Rabbit anti ly6g, by Abcam (1 mentions)
Alexa flour 488 conjugated, by Thermo Fisher Scientific (1 mentions)
Af2535, by R&D Systems (1 mentions)
Ab240946, by Abcam (1 mentions)
Ab86379, by Abcam (1 mentions)
Ab64693, by Abcam (1 mentions)
Af591, by R&D Systems (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
H 1200, by Vector Laboratories (1 mentions)
6 protocols using ab25235
1
Aortic Macrophage Immunofluorescence Staining
2
Immunohistochemical Staining of Brain Sections
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Immunohistochemical staining was performed as previously described (Miao et al. 2020 (link)). Briefly, coronal brain sections were blocked with 5% goat serum in phosphate-buffered saline with 0.1% Triton-X 100 for 1 h, followed by primary antibody incubations for 1 h at room temperature and overnight incubation at 4 °C. The following primary antibodies were used: Rat anti MAP2 (1:400; Millipore), Rabbit anti Ly6G (1:300; Abcam), Rabbit anti F4/80 (1:300; Bio Legend), Alexa Flour 488-conjugated (1:300; Invitrogen), Rat anti CD31 (1:300; BD Biosciences). Rabbit anti-MMP-2 (1:200, ab97779, Abcam), goat anti-CD206 (1:250, AF2535, R&D), rat anti-CD16/32 (1:250, ab25235, Abcam), and rabbit anti-Iba1 (1:1000, ab5076, Abcam).
3
Immunofluorescence Analysis of Kidney Macrophages
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Paraffin-embedded kidney tissues were cut into 5-μm-thick slices. The slices were dewaxed, hydrated, washed with PBS twice for 5 min each, and then incubated with a 3% H2O2 methanol solution at room temperature for 10 min after repair. The slices were incubated with an F4/80 monoclonal antibody (1:1000, ab240946, Abcam, USA), a CD16 monoclonal antibody (1:1000, ab25235, Abcam, USA), a CD206 monoclonal antibody (1:1000, No.141707, BD Pharmingen, USA) or PBS (the negative control) overnight at 4 °C. Then, the slices were incubated with fluorescein isothiocyanate (FITC)-labeled rat anti-goat IgG (1:1000) at 37 °C in the dark for one hour. Subsequently, the slices were sealed with glycerol and visualized under a fluorescence microscope. The FITC-labeled F4/80, CD16 and CD206 indirectly emitted blue-green light.
4
Microglia Activation Marker CD16/32 Antibody
5
Visualizing Synovial Nerve Fibers and Macrophages
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Synovial tissues and DRGs were dissected and immersed into 4% paraformaldehyde in PBS for 4 h. Then explants were embedded in O.C.T. compound (Tissue-Tek, 4583) frozen next to a cryoprotection in a solution of 20% sucrose overnight in room temperature. For immunofluorescent staining, the ankle joint specimens were sagittally sectioned to obtain free-floating 50-μm-thick slices for a better vision of nerve fibers in synovial tissue of ankle joint, while DRG explants were sectioned to regular 3.5-μm-thick slices and both stained for β-III-Tubulin (Cell Signaling Technology, D71G9, 1:400), sFRP2 (Abcam, ab86379, 1:100) in Eppendorf tube. For staining of M1/M2 macrophages in synovial tissue, 3.5-μm-thick slices were obtained for CD16/32 (Abcam, ab25235, 1:20), CD206 (Abcam, ab64693, 1:100) and hematoxylin and eosin staining.
Zeiss LSM710 confocal microscope was used for confocal imaging of samples at an original magnification ×40 and Zen software.
Zeiss LSM710 confocal microscope was used for confocal imaging of samples at an original magnification ×40 and Zen software.
6
Microglial Phenotyping Post-Traumatic Brain Injury
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Mice were anesthetized and transcardially perfused with 0.1 mmol PBS and 4% paraformaldehyde (PFA) at 3 d post-TBI. 20 µm coronal cryosections were permeabilized and incubated in 5% Donkey Serum for 1 h for blocking. Then the brain tissues were incubated in primary antibody overnight at 4 °C. The primary antibody information is as following: anti-Mer antibody (1:400, AF591, R&D Systems), anti-CD16/32 antibody (1:200, Abcam, ab25235), anti-CD206 antibody (1:400, Abcam ab64693), anti-Iba-1 antibody (1:500, Wako, 019-19741) and (1:500, Abcam, ab5076). After the incubation overnight, the cryosections were incubated with the secondary antibodies (1:500, Jackson Immunoresearch Laboratories) for 1 h at room temperature. After that, the sections were rinsed with PBS and covered with fluorescence mounting medium with 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, H-1200).
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