Histological and immunohistochemical analyses were performed as previously described2 (link). The following antibodies were used: Cdh1 (rat, Novex), Cortactin (rabbit, Abcam), GFP (goat, Abcam), GFP (mouse, Roche), ItgA2 (rabbit, Abcam), ItgA6 (rabbit, Abcam), ItgB1 (rabbit, Abcam), Ki67 (rat, Biolegend), Krt19 TROMA-III (rat, DSHB), MLC2 pSer19 (rabbit, NEB), Myosin (rabbit, Abcam), Nestin (mouse, BD transduction), Pdgfrβ (rabbit, NEB), PTK2 pTyr397 (rabbit, ThermoFisher), SMA (mouse, Agilent), SMA (mouse, Sigma), Tomato (rabbit, Rockland), Tomato (goat, Biorbyt), Vimentin (rabbit, NEB), Vinculin (mouse, Sigma-Aldrich). DBA-rhodamine and DBA-FITC were from Vectorlabs. F-actin was stained with Phalloidin-TRITC (Sigma-Aldrich) and nuclei with DAPI (Sigma-Aldrich). F-actin staining of LSL-KrasG12D; p53 F/F; Pdx1-Cre pancreata and wildtype littermate control pancreata was performed on cryosections. Samples were embedded fresh in OCT medium and after sectioning fixed in 5% NBF for 10 min. Slides were washed in 0.2% Triton X-100 in PBS for 10 min and incubated in FLASH blocking buffer for 30 min. Staining reagent incubations were performed as above. Fluorescent stainings were imaged on a Zeiss LSM 780 confocal microscope. Chromogenic DAB stainings were imaged on a Zeiss Axio Scan Z1 Slide Scanner.
Nestin
Manufactured by BD
Sourced in United States, United Kingdom, Canada
Nestin is a laboratory equipment product designed for cell culture applications. It is a high-quality incubator that provides a controlled environment for the growth and maintenance of cells. The device regulates temperature, humidity, and CO2 levels to create optimum conditions for cell culture.
Automatically generated - may contain errors
Lab products found in correlation
Olig2, by Merck Group (7 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (6 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (6 mentions)
Cleaved caspase 3, by Cell Signaling Technology (6 mentions)
Neuronal nuclei (neun), by Merck Group (6 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Cryostat, by Leica (3 mentions)
Vinculin, by Merck Group (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Fv1000, by Zeiss (2 mentions)
Fluoview fv1000, by Zeiss (2 mentions)
Sm2010r, by Leica (2 mentions)
Glial fibrillary acidic protein (gfap), by Fujifilm (2 mentions)
Lsm 800, by Zeiss (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Total erk1 2, by Cell Signaling Technology (2 mentions)
Mcl 1, by Santa Cruz Biotechnology (2 mentions)
Fbxw7, by Abcam (2 mentions)
43 protocols using nestin
1
Comprehensive Tissue Analysis Protocol
2
Characterizing GBM Tumorsphere Phenotypes
3
Characterization of MSC and NSC Markers
4
Immunohistochemical Profiling of Neural Markers
5
Immunostaining of Neural Cell Cultures
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Cells (~ 1 × 105) were cultured on a cover glass in a 12‐well plate with 700 μL of medium. The neural cells were allowed to grow to desired morphology and density before staining procedure. Cells were first washed once with PBS and fixed by 4% paraformaldehyde/4% sucrose in PBS at room temp, followed by permeabilization and DNA denaturation by 0.2% TritonX‐100 in 4 m HCl. After that, the cells were washed with PBS and blocked in 80 μL BSA (3%). The cells were incubated with anti‐FOXG1 (ab18259; Abcam), Ki‐67 (BD, 550609), Otx1/2 (ab21990; Abcam, Cambridge, MA, USA), PAX6 (ab195045; Abcam), NESTIN (BD, 561230), Nkx2.1 (MAB5460; Millipore, Darmstadt, Germany), MAP2 (M4403; Sigma, St. Louis, MO, USA), GABA (A2052; Sigma) VGAT (131011; Synaptic systems, Goettingen, Germany), SYNAPSIN (Abcam), TBR1 (ab31940; Abcam), GAT1 and Glutamate (ab1511; Millipore) in BSA (3%) at 4 °C overnight, and then conjugated with and Hoechst 33342 or DAPI. The glass slides were mounted with a cover slip before imaging.
6
Multimarker Immunohistochemistry for Tissue Analysis
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Tissues were fixed in 4% PBS-buffered paraformaldehyde overnight and then transferred to 30% sucrose (w/v) for another 24 hours. Tissue was then embedded in Optimal Cutting Temperature (OCT) compound and sectioned at 8 microns on a Leica cryostat. Sections were blocked (PBS, 5% goat serum, 0.1% TritonX-100), incubated with primary antibodies for 2 hrs at room temperature in humidity chamber, washed with PBS and incubated with secondary antibodies for 45 min room temperature. Primary antibodies used: GFP (1∶250; Aves Labs Cat# GFP-1020 RRID:AB_10000240), Gabra6 (1∶100; EMD Millipore Cat# AB5610 RRID:AB_91935), NeuN Clone A60 (1∶100; Millipore Cat# MAB377 RRID:AB_2298772), BLBP (1∶250; EMD Millipore Cat# ABN14 RRID:AB_10000325), Calb1 D28K (1∶100; Sigma-Aldrich Cat# C9848 RRID:AB_476894), Olig2 (1∶150; EMD Millipore Cat# AB9610 RRID:AB_570666), Ki67 (1∶100; Abcam Cat# ab15580 RRID:AB_443209), Rspo1 (1∶1000; Sigma-Aldrich Cat# SAB3500046 RRID:AB_10602510), Pax6 (1∶250; Covance Cat# PRB-278P), Prominin-1 (1∶100; eBioscience Cat# 14-1331-90), Nestin (1∶300; BD Biosciences Cat#556309). Species-specific Alexa-Fluor-conjugated secondary antibodies were used for detection (1∶250; Invitrogen). tdTomato expression was visualized directly.
7
Immunocytochemical Staining of Neural Markers
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Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature, permeabilized, and blocked in a blocking solution (10% normal goat serum, Pel-Freez, Rogers, AR, USA), 0.3% Triton X-100 (Sigma-Aldrich in PBS) for 1 h. The samples were stained with primary antibodies for 1 day and then incubated for 1 h with secondary antibodies in the dark. Cells were photographed using an epifluorescence microscope (Leica Micro systems, Wetzlar, Germany). Monoclonal primary antibodies used were as follows: NESTIN (BD Bioscience, Franklin Lakes, NJ), microtubule-associated protein 2 (MAP2; Sigma-Aldrich). Polyclonal primary antibodies used included those against SOX2 (Chemicon, Temecula, CA) and tyrosine hydroxylase (TH; Pel-Freez, Rogers, AR, USA).
8
Quantifying Immune Cell and Vascular Markers
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Immunohistochemistry was performed on 4-μm-thick paraffinembedded tissue sections. Tissue was deparaffinized and 10 mmol/L citric acid, pH6 at 95 °C was used for antigen retrieval. Sections were permeabilized with 0.1% triton/PBS. Antibodies against F4/80 (AbD Serotec), CD68 (Abcam), and CD3 (Dako) were used. The F4/80, CD3, and CD68 positive area was quantified in at least 10 photographs at 200× magnification per animal, using publicly available image processing software (ImageJ; Fiji) and expressed as percentage of the total tissue area. Immunofluorescence was performed on 4 μm-thick frozen sections fixed in methanol. CD146 (homemade rat antimouse), CD31 (Abcam), Claudin-1 (Thermo Scientific), Nestin (BD Pharmingen), Nephrin (Abcam) anti-rabbit, and α smooth muscle actin (α-SMA) (Sigma-Aldrich) anti-mouse antibodies were used. Alexa fluor (Invitrogen) secondary antibodies were used for detection. Images were obtained with an OlympusIX83 photonic microscope at X400 magnification.
9
Differentiation of MPS IIIB Neurons
10
Immunofluorescence Labeling of Neural Markers
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Labeling for EdU and differentiation markers was carried out as previously described (Corns et al., 2015) . Immunofluorescence was performed with antibodies against DBI (rabbit, 1:2000, Frontier Institute, Japan), ODN (rabbit, 1:250, gift of Dr M. C. Tonon, Universitè of Rouen, France), Nestin (rat, 1:1000, BD Biosciences, Oxford, UK), CD24-FITC (mouse, 1:500, BD Biosciences), NeuN (mouse, 1:1000, Millipore, Watford, UK), Sox2, (goat, 1:1000, Santa Cruz), PanQKI, (mouse, 1:100, UC Davis/NIH Neuromab Facility, Davis, CA), S100β (rabbit, 1:750, Abcam, Cambridge, UK), glial fibrillary acidic protein (GFAP) (mouse, 1:100, Neuromab). All antibodies were diluted in PBS containing 0.1% triton (PBST) except for CD24 which was diluted in PBS. Antibodies were detected using appropriate Alexa 488 or Alexa 555 conjugated secondary antibodies (1:1000 in PBS) (Invitrogen). Sections were mounted on glass slides using vectashield plus DAPI (4',6-diamidino-2-phenylindole) (Vector labs, Peterborough, UK).
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