Cells were cultured on a coated 13 mm glass coverslip (PDL and laminin, as aforementioned) for 48 h in a complete embryonic NeuroCult™ proliferation medium. The cells were washed with PBS, fixed with 4% paraformaldehyde (MERCK #30525-89-4) in PBS for 20 min at room temperature (RT), and subsequently permeabilized with PBS containing 0.5% Triton X-100 for 5 min. Then, the cells were incubated with a blocking solution containing 10% goat serum and 0.2% BSA (Biological Industries Israel Beit Haemek #030-010-1B) in PBS with 0.1% Triton X-100, for 1 h at RT, followed by primary antibody incubation with anti-Nestin (anti-rat 1:250, abcam ab81462) at 4 °C overnight. Next, secondary antibody donkey anti-rat 488 (1:500, abcam ab150153) was incubated for 1 h at RT. Washing with 0.1% Triton X-100 in PBS preceded each step. Finally, coverslips were mounted in Fluoroshield™ Histology Mounting Medium with DAPI (Sigma-Aldrich #F6057). The representative images were taken with a Nikon Eclipse Ti2 wide-field fluorescence microscope (Fig. 1C ).
Ab81462
Manufactured by Abcam
Sourced in United States
Ab81462 is a lab equipment product. It is a recombinant antibody that recognizes and binds to a specific target.
Automatically generated - may contain errors
Lab products found in correlation
Ab150153, by Abcam (1 mentions)
Fluoroshield with dapi histology mounting medium, by Merck Group (1 mentions)
Ab5804, by Merck Group (1 mentions)
Sc 365823, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Cryostar nx70, by Thermo Fisher Scientific (1 mentions)
Neg 50tm frozen section medium, by Thermo Fisher Scientific (1 mentions)
Mms 435p, by BioLegend (1 mentions)
Confocal laser scanning microscopy, by Leica (1 mentions)
Ab97959, by Abcam (1 mentions)
Sc 271430, by Santa Cruz Biotechnology (1 mentions)
Mab3402, by Merck Group (1 mentions)
Nb100 1028, by Novus Biologicals (1 mentions)
4 protocols using ab81462
1
Nestin Expression in Neural Progenitors
2
Immunocytochemical Characterization of NSC Cells
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PFA-fixed NSC cells were washed three times with PBS for 5 min. Then, they were incubated with 0.3% Triton X-100 for 10 min and blocked with 5% bovine serum albumin for 1 h at room temperature. Afterwards, cells were incubated with primary antibodies at 4 °C overnight. The primary antibodies were used as follows: anti-GFAP antibody 1:100 (AB5804; Millipore Corporation, Danvers, MA, USA), anti-nestin (1:100, ab81462, Abcam, Cambridge, MA, USA), anti-MAP2 (1:200, MAB3418), and anti-SOX-2 (1:200 sc-365823, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After rinsing the samples with PBS, the samples were then incubated with corresponding secondary antibodies for 1 h at room temperature. Nuclei were stained using 4,6-diamidino-2-phenylindole (DAPI, 1:1000; Life Technologies, Mulgrave, VIC, Australia).
3
Immunohistochemical Analysis of Organoids
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We immuno-stained 10 µm-thick sections of the sample obtained with a cryostat (Thermofisher, Cryostar nx70). After imaging, samples were fixed in 4% formaldehyde, incubated overnight in 30% sucrose and immersed in pre-warmed embedding solution (7.5% gelatin and 10% sucrose in DI water). Blocks containing multiple organoids were cut out and frozen in − 50 °C isopentane bath. With confocal microscopy (LS980 Zeiss, 40x) we could detect the presence of SOX-2 (ThermoFisher, Invitrogen, eBioscience™, 14-9811-82) and Nestin (Abcam, ab81462) (Fig. 3 , Suppl. Fig. S2 ) within the structures. For TUBB3 (Fig. 3 , Suppl. Fig. S2 ) and N-CAD (Suppl. Fig. S2 ) staining, samples were embedded and cryosectioned directly in Neg-50TM Frozen Section Medium (Thermo Fisher Scientific, 6502B) and stained with anti-N-Cadherin antibody (ThermoFisher, Invitrogen, PA5-29570) and anti-Tubulin ß 3 antibody (Clone Tuj1, BioLegend, MMS-435P) respectively.
4
Immunohistochemical Analysis of Brain Cells
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The sample collection procedure was the same as that for brain atrophy volume measurement. Thereafter, cells or brain sections were fixed in 4% PFA for 10 min, permeabilized in 0.3% Triton X-100 for 10 min, and then incubated in 10% bovine serum albumin (BSA) for 1 h at room temperature to block non-specific binding. Finally, they were incubated overnight at 4 °C with primary antibodies against the following as appropriate: GFAP (1:500; MAB3402, Millipore, Billerica, MA), BrdU (1:200; sc-56255, Santa Cruz, CA), Iba-1 (1:200; NB100-1028, Novusbio, Lieeleton), arginase (1:500; sc-271430, Santa Cruz, CA), CD206 (1:500, R&D System, AF2535), nestin (1:500, ab81462, Abcam, Cambridge, MA), and Sox2 (1:500, ab97959, Abcam, Cambridge, MA). For BrdU staining, brain sections were incubated with 2 M hydrochloric acid at 37 °C for 30 min and neutralized with 0.1 M sodium borate (pH 8.5) for 10 min after permeabilization and before blocking. After removing excess primary antibodies, the samples were incubated with fluorescence-tagged secondary antibodies at room temperature for 1 h. Excess secondary antibodies were removed, and the samples were incubated with 4, 6-diamidino-2-phenylindole (DAPI, BIOTIUM-23002, Life Technologies) for 10 min at room temperature. The samples were then imaged using laser scanning confocal microscopy (Leica, Soloms, Germany) 27 (link).
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