Immunofluorescent staining methods were used to detect the expression of Akt, Ki67, Tuj-1, and GFAP and BrdU incorporation in this study using anti-Akt (05-591, Millipore), anti-Ki67 (AB9260, Millipore), anti-Tuj-1 (MAB5564, Millipore), anti-GFAP (AB5804, Millipore), and anti-BrdU (MAB4072, Millipore) antibodies. Specifically, cultures or sliced neurosphere sections (10 μm) were fixed in 4% PFA for 30 min, placed in blocking buffer (5% goat serum) for 40 min and incubated in diluted primary antibodies (dilution prepared in PBS containing 5% BSA (BP1600-100, Fisher BioReagents)) overnight at 4 ºC. Cultures were rinsed in PBST (PBS containing 1% Triton X-100 (BP151, Fisher BioReagents)) three times for 10 min for each. Then, samples were incubated in secondary antibodies (Rabbit IgG Alexa 594/ Mouse IgG Alexa 488, Invitrogen) for 1 hour at room temperature followed by DAPI incubation (1 μg/ml) for 10 min at room temperature. Finally, cultures were rinsed with PBST three times for 10 min for each, and mounted in aqueous mounting medium (ab128982, Abcam). Images of the slides were captured using an Olympus BX60 upright fluorescent microscope (Olympus Inc., Japan) with Hamamatsu imaging system (Hamamatsu C4742-95 camera and Imaging program-HCImage 2.1 Live Version, Hamamatsu Photonics Inc., Japan).
Ab128982
Manufactured by Abcam
Sourced in United Kingdom
Ab128982 is a monoclonal antibody that recognizes the protein Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH is a key enzyme involved in glycolysis, the metabolic pathway that converts glucose into energy. The antibody can be used for the detection and quantification of GAPDH in various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Ab5804, by Merck Group (1 mentions)
Mab4072, by Merck Group (1 mentions)
Ab9260, by Merck Group (1 mentions)
Bp151, by Thermo Fisher Scientific (1 mentions)
Mab5564, by Merck Group (1 mentions)
Bp1600 100, by Thermo Fisher Scientific (1 mentions)
2 protocols using ab128982
1
Immunofluorescent Staining of Neural Markers
2
Immunofluorescence Staining of Cultured Cells
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Cells were cultured on poly-L-lysine coated glass coverslips until around 80% confluence. They were washed with DPBS and then fixed with 4% paraformaldehyde for 10 min. After washing with ice-cold phosphate buffered saline (PBS) for 3 times, they were permeabilized with 0.1% Triton X-100 in PBS for 10 min and washed with PBS 3 times for 5 min each. The cells were blocked with 1% bovine serum albumin (BSA) in PBST for 30 min. Incubation of primary antibodies (Table 1 ) was done in 1% BSA at 4 °C overnight, followed by washes with PBS, 3 times for 5 min each. Secondary antibody incubation was done in 1% BSA for 1 h at room temperature and washed with PBS 3 times for 5 min each. Nuclear staining was done with Hoechst 33,342 (ThermoFisher, H3570, Waltham, MA, USA) at 1:1500 dilution in PBS for 5 min. The cells were washed with PBS and mounted in aqueous mounting medium (Abcam, ab128982, Cambridge, UK) for imaging.
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