RPCs were plated on glass coverslips (VWR, West Chester, PA, USA) coated with laminin (Sigma-Aldrich, Saint Louis, MO, USA) in 24-well plates. Following transfection with miR-29a mimic, miR-29a inhibitor, siRbm8a or Rbm8a clone, cells were fixed with 4% paraformaldehyde (Sigma- Aldrich), and permeabilized with 1% Triton X-100 (Invitrogen) [46 (link)]. Then, The cells were incubated with an optimal concentration of rabbit polyclonal anti-RBM8A antibody (Novus; 1:100), mouse monoclonal anti-nestin (BD, San Jose, CA, USA; 1:200), mouse monoclonal anti-Ki-67 (BD; 1:200), mouse monoclonal anti-β3-tubulin (Chemicon, Billerica, MA, USA; 1:100), mouse monoclonal anti-Rhodopsin (Chemicon; 1:100) or mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (Chemicon; 1:200) overnight at 4°C followed by an incubation with fluorescently labeled secondary antibodies (Alexa Fluor546-goat anti-mouse/rabbit, BD, San Jose, CA, USA; 1:800). After rinsing three times with PBS, nuclei were stained with 4′,6-diamidino-2-phenylindole (Hoechst; Invitrogen, Molecular Probes). Negative control samples were processed in parallel but without the primary antibody. Immunoreactive cells were visualized, and images were recorded using a fluorescence microscope (Olympus BX51, Japan).
Mouse monoclonal anti nestin
Manufactured by BD
Sourced in United States
Mouse monoclonal anti-nestin is a primary antibody that specifically binds to the nestin protein, an intermediate filament protein expressed in neural stem and progenitor cells.
Automatically generated - may contain errors
Lab products found in correlation
Glass coverslip, by Avantor (1 mentions)
Laminin, by Merck Group (1 mentions)
Mouse monoclonal anti ki 67, by BD (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Goat polyclonal anti gfp, by Abcam (1 mentions)
Chicken polyclonal anti gfp, by Aves Labs (1 mentions)
Mouse monoclonal anti pax6, by Developmental Studies Hybridoma Bank (1 mentions)
Odyssey v3.0 image scanning, by LI COR (1 mentions)
0.22 mm polyvinylidene fluoride membranes, by Merck Group (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Merck Group (1 mentions)
Mouse monoclonal anti neun, by Merck Group (1 mentions)
Rat monoclonal anti brdu, by Abcam (1 mentions)
Alexa fluor 488 labeled donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Confocal laser scanning microscope, by Olympus (1 mentions)
4 protocols using mouse monoclonal anti nestin
1
Immunofluorescence Analysis of Rbm8a in RPCs
2
Immunofluorescence Staining Protocol
3
Quantitative Protein Analysis of miRNA-Transfected RPCs
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After the transfection of miRNAs into RPCs, the total proteins of the cells were harvested at specified time points. A BCA kit (Pierce, Rockford, IL, USA) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to analyze the protein concentrations and separate the proteins, respectively24 (link). Following SDS-PAGE, the proteins were transferred to 0.22 mm polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Next, the membranes were blocked with 5% nonfat milk, and they were then incubated with rabbit polyclonal anti-TLX (Santa Cruz Biotechnology, 1:200), mouse monoclonal anti-nestin (BD, 1:500), and mouse anti-β-actin (Sigma, 1:0000) at 37°C for 2 hours. The membranes were next incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma, 1:5000). Protein expression images were visualized using Odyssey V 3.0 image scanning (LI-COR, Lincoln, NE, USA). Quantification of the densitometric intensities of the protein bands was performed using the Bandscan 5.0 software, and the values were normalized against β-actin for each sample.
4
Immunohistochemical Analysis of Neurogenesis
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Frozen 30-μm-thick sections were incubated in blocking solution (10% normal donkey serum or 10% normal goat serum and 0.3% Triton X-100 in PBS, pH 7.5) for 2 h at room temperature (RT) and then incubated with primary antibodies in 5% serum and PBS for 24 h at 4°C. The sections were subsequently incubated for 3 h at RT with fluorophore-conjugated secondary antibodies. To prepare the sections for BrdU staining, they were incubated in 2 N HCl for 0.5 h at 37°C.
Rat monoclonal anti-BrdU (1:100; Abcam, UK) primary antibodies were used to mark proliferating cells, mouse monoclonal anti-Nestin (1:100; BD Pharmingen, USA) primary antibodies were used as a marker for NSCs, and mouse monoclonal anti-NeuN (1:100; Chemicon, USA) primary antibodies were used to label mature neurons. The following secondary antibodies were used: Alexa Fluor 594-labeled donkey anti-rat IgG and Alexa Fluor 488-labeled donkey anti-mouse IgG (1:200 for both; Invitrogen, USA).
The stained slides were dehydrated, cover-slipped in anti-quenching agent (p-phenylenediamine, PPD) and analyzed using a confocal laser-scanning microscope (Olympus, Tokyo, Japan). The number of positive cells was counted in a blinded manner in the ipsilateral SGZ using 20X and 40X objectives in OLYMPUS FV10-ASW Viewer.
Rat monoclonal anti-BrdU (1:100; Abcam, UK) primary antibodies were used to mark proliferating cells, mouse monoclonal anti-Nestin (1:100; BD Pharmingen, USA) primary antibodies were used as a marker for NSCs, and mouse monoclonal anti-NeuN (1:100; Chemicon, USA) primary antibodies were used to label mature neurons. The following secondary antibodies were used: Alexa Fluor 594-labeled donkey anti-rat IgG and Alexa Fluor 488-labeled donkey anti-mouse IgG (1:200 for both; Invitrogen, USA).
The stained slides were dehydrated, cover-slipped in anti-quenching agent (p-phenylenediamine, PPD) and analyzed using a confocal laser-scanning microscope (Olympus, Tokyo, Japan). The number of positive cells was counted in a blinded manner in the ipsilateral SGZ using 20X and 40X objectives in OLYMPUS FV10-ASW Viewer.
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