The treated mouse was transcranial perfused with 15 ml of 1×phosphate‐buffered saline. Harvested the brain and gently separated it into small pieces. The segments were transferred into a 50 ml tube, 10 ml of trypsin was added, and incubated at 37 °C for 10 min. After incubation, triturated and dissociated the brain clumps, and then added 25 ml RPMI1640 culture medium (Sigma‐Aldrich, St. Louis, USA). Filtered the suspension through a 60 μm filter and span at 400 g for 5 min. Resuspend cells with RPMI1640 culture medium and diluted cells to a concentration of 5 × 105 cells/ml. Brain cells were sorted by CytoFLEX S cytometer (Beckmann Coulter, Pasadena, USA) and analyzed by FlowJo software (BD Biosciences). Microglia were identified by CD68 (MCA341, Serotec), oligodendrocytes were identified by OSP (Ab7474, Abcam), astrocytes were identified by GFAP (G3893, Sigma‐Aldrich), and the rest of the cells were regarded as neurons. Cellular reactive oxygen species (ROS) of microglia were measured using the dihydroethidium probe as previously described.26
Ab7474
Manufactured by Abcam
Ab7474 is a laboratory equipment product. It is designed for general laboratory use. The core function of this product is to assist in various scientific experiments and procedures.
Automatically generated - may contain errors
Lab products found in correlation
Glial fibrillary acidic protein (gfap), by Merck Group (1 mentions)
Rpmi 1640 culture medium, by Merck Group (1 mentions)
Cytoflex s cytometer, by Beckman Coulter (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
Casp3, by Abcam (1 mentions)
Ab104224, by Abcam (1 mentions)
Ab8135, by Abcam (1 mentions)
Ab10062, by Abcam (1 mentions)
T8578, by Merck Group (1 mentions)
2 protocols using ab7474
1
Isolation and Characterization of Mouse Brain Cells
2
Histological Analysis of Spinal Cord Injury
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For histological analysis, the spinal cord specimens were managed the same as above reported. Immunofluorescent staining was performed on 20‐μm sections, which were stained with the following antibodies: green fluorescence protein (GFP, 1:200, A10262, Molecular probes); neurofilament (NF, 1:200, Ab8135, Abcam); caspase 3 (Casp3, 1:200, Abcam); neuron‐specific marker (NeuN, 1:200, Ab104224, Abcam), ß‐tubulin III (Tubulin; 1:100, T8578, Sigma); glial fibrillary acidic protein (GFAP, 1:400, Ab10062, Abcam); oligodendrocyte‐specific antibody (O4, 1:200, Ab7474, Abcam); and 4′,6‐diamidino‐2‐phenylindole (DAPI, 1:1000, D1306, Invitrogen). Sections of spinal cord were imaged at 10×, 20×, and 40× magnifications using a confocal microscope (Leica SP5, Germany). GFP staining was performed to check the survival of the BMSCs. NF staining was used to detect axon growth in the injured area. NeuN staining was performed to detect the spared neurons in the area adjacent to the lesion. Double immunofluorescent assay (GFP/Tubulin, GFP/GFAP, GFP/O4) was performed to determine whether the GFP+ cells (include the BMSCs) differentiate into other types of cell.
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