Intervened cells were fixed in 4% paraformaldehyde, penetrated with 0.1% Triton X-100, blocked with 5% bovine serum albumin (BSA) and incubated with primary antibodies at 4°C overnight. Afterwards, the cells were stained with fluorescein-labeled secondary antibodies and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) under dark conditions, sealed on the slides, and observed under a laser confocal microscope. The respective concentrations of primary antibodies were: Iba1 (wako, Lot: 019–19741, 1:1000), CD86 (BD, Lot: 553689, 1:200), iNOS (BD, Lot: 610328, 1:200), CD206 (Santa, sc-34577, 1:100), Arg1 (Santa, sc-18351, 1:100), Nestin (Abcam, ab6142, 1:200), SOX2 (Abcam, ab97597, 1:200), Tuj-1 (Abcam, ab78078, 1:200), Olig2 (Abcam, ab109186, 1:100), GFAP (Abcam, ab7260, 1:1000), MAP2 (Abcam, ab5392, 1:500), O4 (Sigma, O7139, 1:100), A minimum of four images was captured using a 20X objective on a Zeiss microscope (Zeiss AxioCam, Germany) and the positive cells were counted using Image J.
O7139
Manufactured by Merck Group
Sourced in Germany, United Kingdom
O7139 is a laboratory equipment product manufactured by Merck Group. The core function of this product is to facilitate specific laboratory processes, though a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab5392, by Merck Group (1 mentions)
Ab78078, by Abcam (1 mentions)
Axiocam, by Zeiss (1 mentions)
Ab109186, by Abcam (1 mentions)
Ab6142, by Abcam (1 mentions)
Ab7260, by Abcam (1 mentions)
Ab32454, by Abcam (1 mentions)
Lsm 780, by Zeiss (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Ab18207, by Abcam (1 mentions)
Ab150084, by Abcam (1 mentions)
Ab150117, by Abcam (1 mentions)
Ab92391, by Abcam (1 mentions)
Ab91279, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Ab833, by Abcam (1 mentions)
Ab15323, by Abcam (1 mentions)
3 protocols using o7139
1
Immunofluorescence Imaging of Neural Markers
2
Immunocytochemical Characterization of Neural Cell Lineages
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All samples
were fixed with 4% formaldehyde for 20 min and then washed three times
in PBS. Then, the fixed cells were permeabilized with 0.1% Triton-X
in PBS for 5 min and later washed with PBS three times, and the cells
were then blocked with 10% goat serum (Gibco, USA) in PBS for 30 min
at room temperature. After removal of the blocking reagent, the cells
were incubated with primary antibody dilution, including rabbit anti-Nestin
(1:250, ab92391, Abcam), mouse anti-GFAP (1:300, 3670S, CST), rabbit
anti-β-tubulin III (1:1000, ab18207, Abcam), rabbit anti-MAP2
(1:1000, ab32454, Abcam), mouse anti-Oligodencyte Marker O4 (1:50,
O7139, Sigma), and mouse anti-NF (1:800, Abcam) at 4 °C overnight.
The secondary antibodies, goat anti-rabbit IgG H&L (Alexa Fluor
594, 1:200, ab150084, Abcam) and goat anti-mouse IgG H&L (Alexa
Fluor 488, 1:200, ab150117, Abcam), were used to react with the cells
for 1 h at room temperature. Then, the cells were washed with PBS,
and the nucleus was stained with DAPI for 5 min. The samples were
visualized by indirect fluorescence under the fluorescent microscope
(Leica, Germany), confocal fluorescence microscopy (Zeiss LSM 780,
Germany), or fluorescence microscopy Olympus IX81 (Olympus, Japan).
were fixed with 4% formaldehyde for 20 min and then washed three times
in PBS. Then, the fixed cells were permeabilized with 0.1% Triton-X
in PBS for 5 min and later washed with PBS three times, and the cells
were then blocked with 10% goat serum (Gibco, USA) in PBS for 30 min
at room temperature. After removal of the blocking reagent, the cells
were incubated with primary antibody dilution, including rabbit anti-Nestin
(1:250, ab92391, Abcam), mouse anti-GFAP (1:300, 3670S, CST), rabbit
anti-β-tubulin III (1:1000, ab18207, Abcam), rabbit anti-MAP2
(1:1000, ab32454, Abcam), mouse anti-Oligodencyte Marker O4 (1:50,
O7139, Sigma), and mouse anti-NF (1:800, Abcam) at 4 °C overnight.
The secondary antibodies, goat anti-rabbit IgG H&L (Alexa Fluor
594, 1:200, ab150084, Abcam) and goat anti-mouse IgG H&L (Alexa
Fluor 488, 1:200, ab150117, Abcam), were used to react with the cells
for 1 h at room temperature. Then, the cells were washed with PBS,
and the nucleus was stained with DAPI for 5 min. The samples were
visualized by indirect fluorescence under the fluorescent microscope
(Leica, Germany), confocal fluorescence microscopy (Zeiss LSM 780,
Germany), or fluorescence microscopy Olympus IX81 (Olympus, Japan).
3
Immunohistochemical Analysis of Mouse Brain
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After fixing with 4% paraformaldehyde, the mouse brain and optic nerve sections were cut at 15-μm thickness at -20 °C and mounted on glass slides. Then, sections were blocked with 3% bovine serum albumin (BSA) at room temperature for 1 h before incubation with rabbit anti-mouse CC1 (OP80, Merck, Kenilworth, NJ), mouse anti-O4 (O7139, Sigma), rabbit anti-ki67 (ab833, Abcam, Cambridge, UK), rabbit-anti-arg-1 (ab91279, Abcam), mouse anti-Iba-1 (SAB2702364, Sigma), and rabbit anti-inducible nitric oxide synthase (iNOS; ab15323, Abcam) monoclonal antibodies in 1% BSA at 4 °C overnight. Sections were washed three times in PBS, incubated with tetramethylrhodamine (TRITC)-labeled and fluorescein isothiocyanate (FITC)-labeled secondary antibody (Abcam) diluted 1:500 in 1% BSA at 37 °C for 1 h, and washed again in PBS. Then, DAPI (1:1000 dilution, 4083S Beyotime Biotechnology, Haimen, China) was used to stain the nuclei for 5 min. Specimens incubated without a primary antibody was used as negative controls. Slides were observed under a confocal microscope.
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