Cells were seeded in eight-chamber slides, grown until confluence and then fixed with 4% wt/vol. paraformaldehyde for 15 min at room temperature. This step was followed by an incubation overnight at 4°C with the primary antibodies reported in ESM Table 3 : rabbit anti-human CD146 (Abcam), rabbit anti-human CXCL12 (Cell Technologies, Cambridge, UK), rabbit anti-human vasclular endothelial-cadherin (VE-cadherin, Abcam), mouse anti-human nestin (Abcam), rabbit anti-human PDGFRβ (Santa Cruz, Heidelberg, Germany), rabbit-anti-human vascular endothelial growth factor receptor 2 (VEGFR2, Abcam), rabbit LEPROT (Abcam), and mouse VWF (Abcam). All incubations were performed overnight at 4°C in PBS. Then, the cells were washed with PBS and incubated with appropriate secondary antibody diluted 1:200 in PBS for 2 h at room temperature. Finally, cells were washed with PBS and counterstained with DAPI 1 μg/μl, for 1 min at room temperature. Images of random fields were obtained at ×200 magnification. Antibodies were validated previously by using negative and/or positive cell cultures as controls, according to the literature. Negative controls have been performed for each immunohistochemistry and immunocytochemistry experiment omitting the primary antibody.
Mouse anti human nestin
Manufactured by Abcam
Sourced in Germany
Mouse anti-human nestin is a primary antibody that detects the nestin protein, which is a type VI intermediate filament protein. Nestin is a marker for neural stem and progenitor cells. This antibody can be used for the detection of nestin in various applications, such as immunohistochemistry and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Glial fibrillary acidic protein (gfap), by Abcam (2 mentions)
Vectashield antifade mounting media, by Vector Laboratories (2 mentions)
Mouse anti human nuclei, by OriGene (2 mentions)
Mouse anti rat gfap, by Merck Group (2 mentions)
Rabbit anti human pdgfrβ, by Santa Cruz Biotechnology (1 mentions)
Vegfr2, by Abcam (1 mentions)
Rabbit anti human cd146, by Abcam (1 mentions)
3 protocols using mouse anti human nestin
1
Immunocytochemistry Analysis of Cell Markers
2
Immunohistochemical Characterization of Brain Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Following 90d, animals were euthanized, perfused transcardially and their brains removed and post-fixed. Serial 30μm sections (every 10th section) were stained (0.1% cresyl violet acetate). Prussian blue staining for SPIO containing cells was performed (17 (link)).
Free-floating sections were blocked with 10% normal goat serum (NGS; Invitrogen, Carlsbad, CA) for 10 min. Primary antibodies were diluted in 0.1M PBS containing 10% NGS and 0.1% triton x-100 and used at the following concentrations: rabbit anti-human glial fibrillary acidic protein 1:200 (GFAP; Abcam, Cambridge, MA); mouse anti-human nestin 1:100 (Abcam); mouse anti-human cyclic nucleotide phosphodiesterase 1:100 (CNPase; Millipore, Temecula, CA); mouse anti-human nuclei 1:100 (Acris Antibodies, Germany); and mouse anti-rat GFAP 1:300 (Sigma-Aldrich, St Louis, MO). Secondary antibodies (1:1000; Invitrogen) included: goat anti-rabbit AlexaFluor (488 or 568 nm) or goat anti-mouse AlexaFluor (488 or 555 nm). Sections were air-dried and coverslipped with VectaShield anti-fade mounting media (Vector Labs, Burlingame, CA). Slides were stored at 4°C and immunolabeled slides were scanned on a confocal microscope (BioRad 1024). Z-series of 10–12 images were collected by stepping through ~1μm sections for each tissue.
Free-floating sections were blocked with 10% normal goat serum (NGS; Invitrogen, Carlsbad, CA) for 10 min. Primary antibodies were diluted in 0.1M PBS containing 10% NGS and 0.1% triton x-100 and used at the following concentrations: rabbit anti-human glial fibrillary acidic protein 1:200 (GFAP; Abcam, Cambridge, MA); mouse anti-human nestin 1:100 (Abcam); mouse anti-human cyclic nucleotide phosphodiesterase 1:100 (CNPase; Millipore, Temecula, CA); mouse anti-human nuclei 1:100 (Acris Antibodies, Germany); and mouse anti-rat GFAP 1:300 (Sigma-Aldrich, St Louis, MO). Secondary antibodies (1:1000; Invitrogen) included: goat anti-rabbit AlexaFluor (488 or 568 nm) or goat anti-mouse AlexaFluor (488 or 555 nm). Sections were air-dried and coverslipped with VectaShield anti-fade mounting media (Vector Labs, Burlingame, CA). Slides were stored at 4°C and immunolabeled slides were scanned on a confocal microscope (BioRad 1024). Z-series of 10–12 images were collected by stepping through ~1μm sections for each tissue.
3
Immunohistochemical Characterization of Brain Tissue
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