Lyophilized human fibrinogen and Millex syringe filter were purchased from EMD Millipore (Billerica, MA). Lyophilized bovine thrombin, calcium chloride and ε-aminocaproic acid (εACA) were purchased from Sigma-Aldrich (St. Louis, MO). Sulfosuccinimidyl 6-(3’-(2-pyridyldithio)propionamido)hexanoate (Sulfo-LC-SPDP) was purchased from Thermo Fisher Scientific (Newington, NH). Human Factor XIII was purchased from Enzyme Research Laboratories (South Bend, IN). Dialysis membrane was purchased from Spectrum Laboratories (Rancho Dominguez, CA). Picrosirius red staining was purchased from Abcam (Cambridge, MA). TO-PRO-3 iodide, Alexa Fluor 488 conjugated anti-rabbit IgG secondary antibody and Alexa Fluor 568 conjugated anti-mouse IgG secondary antibody were purchased from Invitrogen (Carlsband, CA). Rabbit anti-aquaporin 5, mouse anti-cytokeratin 7, rabbit anti-Ki-67, mouse anti-ICAM-1, rabbit anti-N-cadherin and mouse anti-beta III tubulin were purchased from Abcam. Rabbit anti-VCAM-1 was purchased from Cell Signaling Technology (Danvers, MA). Peptides were synthesized by University of Utah DNA/Peptide synthesis core facility. Female C57BL/6 mice at 6 weeks old weighing 15–19 g were purchased from the Jackson Laboratory (Bar Harbor, ME).
Mouse anti beta 3 tubulin
Manufactured by Abcam
Sourced in United States, United Kingdom
Mouse anti-beta III tubulin is a primary antibody that specifically recognizes the beta III isoform of tubulin, a key structural component of microtubules. This antibody can be used to detect and quantify beta III tubulin expression in various cell and tissue types.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Lyophilized bovine thrombin, by Merck Group (1 mentions)
Picrosirius red staining, by Abcam (1 mentions)
Rabbit anti aquaporin 5, by Abcam (1 mentions)
Mouse anti icam 1, by Abcam (1 mentions)
Rabbit anti vcam 1, by Cell Signaling Technology (1 mentions)
ε aminocaproic acid εaca, by Merck Group (1 mentions)
Sulfosuccinimidyl 6 3 2 pyridyldithio propionamido hexanoate sulfo lc spdp, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 conjugated anti mouse igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Mouse anti cytokeratin 7, by Abcam (1 mentions)
Rabbit anti n cadherin, by Abcam (1 mentions)
Millex syringe filter, by Merck Group (1 mentions)
Rabbit anti ki67, by Abcam (1 mentions)
Calcium chloride, by Merck Group (1 mentions)
Mouse anti gfap, by Merck Group (1 mentions)
Mouse anti γh2ax, by Merck Group (1 mentions)
Ix71 microscope, by Hamamatsu Photonics (1 mentions)
Rabbit anti tbr1, by Abcam (1 mentions)
7 protocols using mouse anti beta 3 tubulin
1
Fibrin Hydrogel Scaffold Characterization
2
Immunodetection of OCT3, Beta-III Tubulin, and GFAP
3
Immunocytochemistry of Neuronal Markers
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The cells were fixed with 4% paraformaldehyde for 15 min. After several washes, they were blocked with 0.01M phosphate-buffered saline supplemented with 1% BSA and 0.3% Triton X-100 for 30 min. Primary antibodies (mouse anti-LAP2α, 1:500, Abcam, UK; mouse anti-betaIII tubulin, 1:500, Abcam; mouse anti-NeuN, 1:1000, Abcam; rabbit anti-NeuN, 1:200, Abways; rabbit anti-Tbr1, 1:500, Abcam; mouse anti-γH2AX, 1:250, Millipore, USA; mouse anti-Lamin A/C, 1:200, Abcam; rabbit anti-H3K9me3, 1:4000, Abcam; mouse anti-6E10, 1:200, Abcam) were diluted with blocking buffer and applied overnight at 4° C. The cells were stained with suitable Alexa Fluor–labeled secondary antibodies (Invitrogen) in blocking buffer at a concentration of 1:500 for 1.5 h at room temperature. 4', 6-Diamidino-2-phenylindole (Thermo Fisher, IL, USA) was applied to counterstain cells for visualizing nuclei at 1:1000 in Milli Q water (Biocel, Millipore, USA). The images were obtained with an Olympus IX71 microscope using a Hamamatsu ORCA CCD camera and Leica TCS SP5.
4
Immunohistochemical Analysis of Neural Markers
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Freshly frozen Brain tissue sections were fixed with 4% paraformaldehyde for 15 min. Afterward, the brain slices were blocked in 5% normal goat serum for 1 h at 37°C followed by the incubation of primary antibodies 4°C overnight. The staining was visualized by labeling the corresponding secondary antibodies and then counter-stained with DAPI (4′,6′-diamidino-2-phenylindole) for 10 min at 37°C. The following antibodies used: mouse anti-beta III Tubulin (1:1,000; Abcam), Alexa Fluor 594 conjugated goat anti-mouse IgG (1:300; Proteintech). All images were collected and analyzed with an LSM780 confocal laser scanning microscope combined with the upright microscope.
5
Immunostaining of Neuronal Cultures
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MN cultures were fixed for 15 min on ice with 4% paraformaldehyde plus 0.1% glutaraldehyde in PBS. The cultures were permeabilized with 0.1% Triton X-100 for 15 min and incubated for 2 h at room temperature (25°C) in blocking solution [0.1% Triton X-100, 2% bovine serum albumin (BSA), and 10% goat serum in PBS]. Primary antibodies diluted in the blocking solution were incubated overnight at 4°C. After washing with PBS, the cultures were further incubated for 1 h at room temperature with the secondary antibodies diluted in the blocking solution. The slides were then washed with PBS, rinsed with distilled water, and mounted with Prolong Antifade mounting kit (Thermo Fisher Scientific). The primary antibody used was mouse anti-beta III tubulin (1:3,000, Abcam, Cambridge, UK, Cat# ab41489, RRID: AB_727049), and the secondary was Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (1:1,000, Thermo Fisher Scientific, # A-11029, RRID: AB_2534088). Nuclei were stained with DAPI. Images were captured with a DC290 Zoom Kodak Digital Camera coupled to a Nikon fluorescence microscope.
6
Immunofluorescence Staining of Neural and Metabolic Markers
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Immunofluorescence staining was performed on frozen sections. In brief, sections were rinsed in PBS and treated with 0.3 % Triton X-100 in PBS to disrupt the membranes. After blocking with 10 % normal goat serum for 1 h to reduce nonspecific staining, sections were incubated with the primary antibodies, mouse anti-beta III tubulin (Abcam, USA, 1:100) and rabbit anti-PGC-1α (Novus, USA, 1:150) overnight at 4 °C for 24 h. After rinsing in PBS, the secondary antibodies, Alexa Fluor 488 goat anti-mouse IgG (H + L) (Jackson, USA, 1:600) and Cy3 goat anti-rabbit IgG (H + L) (Jackson, USA, 1:800), were added onto sections in 5 % blocking solution, to incubate at room temperature for 30 min. Then, the sections were counterstained with DAPI (Sigma, USA), and imaged using a Leica DFC310 FX microscope (Leica, Japan).
7
Multilineage Differentiation of EB-NPCs
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For flow cytometric analysis of EB-NPCs, cells were collected using Accutase and stained with mouse anti-Nestin, mouse anti-human Sox1, and mouse anti-Sox2 per manufacturer’s instructions using the Human Neural Lineage Analysis kit (BD). For immunofluorescence microscopy, EB-NPCs were seeded on Geltrex-coated slides, fixed with 4% paraformaldehyde, and stained with rat anti-Nestin (1:500; Millipore), rabbit anti-Sox2 (1:200; Epitomics), or rabbit anti-Pax 6 (1:50; BioLegend) before addition of respective secondary antibodies (goat anti-rabbit AlexaFluor 568 and goat anti-rat AlexaFluor 488; both from Thermo Fisher). For analysis of multipotency, EB-NPCs were cultured in neuronal differentiation medium (Neurobasal, 1X B27, 1X GlutaMAX), astrocyte differentiation medium (DMEM [Thermo Fisher], 1X N2, 1X GlutaMAX, 1% FBS [Atlanta Biologicals]), or oligodendrocyte differentiation medium (Neurobasal, 1X B27, GlutaMAX, 30 ng/ml T3 [Sigma]) for at least 14 days before being fixed with 4% paraformaldehyde and stained with mouse anti-beta-III-tubulin (1:500; Abcam), rabbit anti-GFAP (1:200; Thermo Fisher), rabbit anti-NG2 (1:200; Chemicon), or rabbit anti-Olig2 (1:100; Abcam). Slides were cover-slipped and mounted using VectaShield Hard Set Mounting Medium with DAPI (Vector Labs), and all images were captured using a Nikon Eclipse Ti inverted microscope.
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