Transduced or mock-treated cells were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X-100/PBS (PBS-Tx), blocked with 10% normal goat serum (NGS) in PBS-Tx and then incubated with primary antibodies in a single antibody staining assay against neuronal tubulin marker MAP2 (1:1,000; Sigma), motor neuron markers Islet-1 (1:1,000; Abcam, Cambridge, UK) or astrocyte marker GFAP (1:1,000; Sigma) diluted in 2% NGS/PBS-Tx overnight at 4 °C. The following day the cells were counter-stained with secondary Alexa-Fluor conjugated antibodies for 1 hour at room temperature, and washed in PBS between the incubations. Finally, coverslips were stained with Hoechst stain (1:2,000; Sigma) to visualize the nuclei and mounted onto a slide using FluoroMount media (Sigma). The images were captured on Leica confocal laser-scanning microscope using 63× oil lens and appropriate optical zoom, and processed using LAS AF Lite and ImageJ softwares.
Islet 1
Manufactured by Abcam
Sourced in United Kingdom
Islet-1 is a transcription factor that plays a crucial role in the development and maintenance of insulin-producing pancreatic islet cells. It is a commonly used marker for the identification and characterization of these cells.
Automatically generated - may contain errors
Lab products found in correlation
Apotome, by Zeiss (4 mentions)
Lsm780 fcs, by Zeiss (2 mentions)
Titin t11, by Bio-Techne (2 mentions)
Ea 53, by GeneTex (2 mentions)
Tubb3, by Fortrea (2 mentions)
Synaptophysin, by Synaptic Systems (2 mentions)
Ankyring, by Merck Group (2 mentions)
Stmn2, by Novus Biologicals (2 mentions)
Smi 32, by BioLegend (2 mentions)
Synapsin, by Merck Group (2 mentions)
Foxp1, by Abcam (2 mentions)
Alexa fluor, by Thermo Fisher Scientific (2 mentions)
Psd95, by Abcam (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Leica confocal laser scanning microscope, by Leica camera (1 mentions)
Fluoromount media, by Merck Group (1 mentions)
Hoechst stain, by Merck Group (1 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (1 mentions)
Synaptophysin, by Abcam (1 mentions)
Calretinin, by Abcam (1 mentions)
8 protocols using islet 1
1
Immunostaining of Neuronal and Glial Markers
2
Immunofluorescence Labeling of Stem Cells
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Cells were immunolabeled as described previously [28 (link)]. Briefly, EBs and cell samples were fixed in 4% paraformaldehyde for 10–15 min, permeabilized with 0.1% Triton X-100/PBS (1X; 140 mM NaCl, 10 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, pH 7.4; Thermo Scientific, Rockford, IL) for 10 min, blocked in 4% bovine serum albumin (BSA) for 30 min, and incubated with primary antibodies overnight at 4 °C. The next day, the samples were washed three times with PBS and subsequently incubated with Alexa Fluor 488 or 555 labeled secondary antibody (1:300, Invitrogen) for 30 min at room temperature in the dark. After washing three times with PBS, the samples were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, 1 μg/ml; Molecular Probes, Carlsbad, CA). Primary antibodies Nanog (Cell Signaling, Danvers, MA), Oct3/4, Pax6, Nestin, Tuj, Chx10 (all from Millipore, Temecula, CA), Sox2, ZO1, Rx, calretinin, iSlet1, synaptophysin (all from Abcam, Burlingame, CA), Otx2 (Invitrogen), Chx10 (Santa Cruz, Dallas, TX), and Rx (Santa Cruz) were used at 1:200 dilution. Pax6 (DSHB, Iowa City, IA) was used at 1:50 dilution. Ki-67 (Abcam) and Brn3b (Abcam) were used at 1:1,000 dilution. Fluorescent confocal images were acquired using a laser scanning microscope (LSM 510; Carl Zeiss, Thornwood, NY).
3
Immunofluorescence Characterization of Stem Cell Markers
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The cultured cells were placed on 12 mm cover slips, fixed in 4% paraformaldehyde (PFA; Sigma) for 10 min at room temperature, washed three times with phosphate-buffered saline (PBS, Geno, China), and treated with a permeabilizing and blocking buffer (10% donkey serum, 0.225% Triton X-100) for 1 hour at room temperature. Then, the cells were incubated with the following primary antibodies: OCT4 (1 : 200, Abcam), Nanog (1 : 250, Abcam), SSEA4 (1 : 250, Abcam), TRA-1-60 (1 : 300, Abcam), SOX1 (1 : 300, Boster), SOX2 (1 : 300, Boster), Nestin (1 : 300, Abcam), Olig2 (1 : 500, Millipore), Pax6 (1 : 100, DSHB), HB9 (1 : 50, DSHB), Islet1 (1 : 250, Abcam), ChAT (1 : 100, Millipore), and TuJ1 (1 : 250, Abcam). All antibodies were diluted in antibody dilution buffer (2% donkey serum, 0.05%Triton X-100), and the cells were incubated with the antibodies overnight at 4°C. After three washing steps, the cells were incubated for 1 hour at room temperature with secondary antibodies: Alexa Fluor (488 or 555) donkey anti-mouse, donkey anti-rabbit, and donkey anti-goat. For the detection of AChR, the cocultured cells were incubated with Alexa Fluor 555-conjugated α-BTX (1 μg/ml, Invitrogen) for 1 hour at 37°C before fixation. The cells were then washed 2 times in PBS and fixed with 4% PFA. All cell samples were observed using an Olympus fluorescence microscope.
4
Immunocytochemistry of Cellular Markers
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Cells were fixed for 20 min at room temperature in 4% paraformaldehyde in PBS. Permeabilization and blocking of nonspecific epitopes was performed simultaneously using 0.1% Triton X-100, 1% BSA and 10% FBS in PBS for 45 min. Subsequently, the primary antibodies (mouse anti tubulin beta 3 (TUBB3) (1:1000, Covance), monoclonal Anti-Myosin (MY-32) (1:500, Sigma), microtubule-associated protein 2 (MAP2) (1:5000, Abcam), neurofilament heavy polypeptide (SMI-32) (1:500, Millipore), choline Acetyltransferase (CHAT) (1:400, Millipore), Islet-1 (1:1500, Abcam), titin (T11) (1:1000, Bio-Techne), sarcomeric alpha Actinin antibody (EA-53) (1:200, GeneTex) were applied overnight at 4 °C in 0.1% BSA in PBS. The next day, the cells were washed with 0.1% BSA in PBS and incubated with the secondary antibody for 1 h at room temperature. Conjugated bungarotoxin (α-Bungarotoxin, CF®640R, biotium, 1:500) was included in the secondary antibody incubation step. Finally, cells were washed three times with 0.1% BSA in PBS-T (0.005% Tween-20), including Hoechst counter-staining for nuclei in the second washing step. Cells were imaged either with a Zeiss ApoTome or a laser scanning confocal microscope (Zeiss LSM780/FCS) and, if necessary, pictures from individual channels were merged using Fiji.
5
Immunocytochemistry of Cellular Markers
6
Immunostaining of neural markers
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Cells were washed once with PBS, fixed with 4% PFA for 20 min, washed again in PBS and blocked for 1 h in 0.1% Triton in PBS with 10% donkey serum. Fixed cells were then washed and incubated overnight with primary antibodies at 4◦C. Primary antibody solution was washed and cells were subsequently incubated with secondary antibodies (1:2000, Alexa Fluor, Life Technologies) at room temperature for 1 h, washed with PBS and stained with DAPI. Primary antibodies used: Tuj1 (R&D, MAB1195), Islet1 (Abcam, ab178400), MAP2 (Abcam, ab5392), Synapsin (Millipore, AB1543), SMI-32 (BioLegend, 801,702), Chat (Millipore, AB144P), Foxp1 (Abcam, ab16645), AnkyrinG (Millipore, MABN466), Synaptophysin (Synaptic Systems, 101,004), PSD-95 (Abcam, ab2723), STMN2 (Novus NBP49461). Images were analyzed using FIJI.
7
Immunostaining of neural markers
8
Intranuclear RNA Foci Detection in DM2
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Intranuclear foci containing (CCUG)exp RNA were dectected in hiPSCs and NPs from DM2 patients using via RNA fluorescence in situ hybridization (FISH) using Cy3-labeled (CAGG)10 DNA probe (Bonifazi et al., 2006 (link)). The cells have been fixed with 4% paraformaldehyde (PFA), permeabilized using 0.3% Triton 100-X, then blocked with 3% BSA and incubated with primary antibodies OCT4, TUJ1, MAP2, GFAP, ISLET1 (Abcam, Cambridge, United Kingdom), LIM3 (Millipore, Burlington, NJ, United States), and MBNL1 (3A4, Santa Cruz Biotechnology, Dallas, TX, United States) labeled with AlexaFluor 488 or 568 secondary antibody. Nuclei have been counterstained with 4,6- diamidino-2-phenylindole. Samples have been observed by confocal microscopy (LEICA TCS SP5). Up to 50 serial sections (each section about 1 μm in thickness), taken from the top to the bottom along the Z axis, have been used to obtain a 3D reconstruction of each analyzed field.
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