Reagent and antibody sources were as follows: AG1478 (Calbiochem/Merck, Darmstadt, Germany), BMP4 (R&D Systems, Minneapolis, MN, USA), DAPI (4′,6-diamidino-2-phenylindole dihydrochloride), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), temozolomide (TMZ) and anti-β-Actin-peroxidase conjugated antibody (Sigma-Aldrich, Munich, Germany), anti-AKT, anti-phospho-AKT (Ser473), anti-phospho-AKT (Thr308), anti-BIM, anti-cleaved caspase 3, anti-cleaved caspase 7, anti-cleaved PARP (poly (ADP-ribose) polymerase-1), anti-EGF Receptor, anti-phospho-EGF receptor (Tyr1068), anti-FOXO3a, anti-phospho-FOXO3a (Thr32), anti-phospho-FOXO3a (Ser253), anti-phospho-FOXO3a (Ser318/321), anti-phospho-Rb (Ser807/811), anti-SMAD1, anti-SMAD3, anti-SMAD4, anti-SMAD5, anti-phospho-SMAD1/5 (Ser463/465), anti-phospho-SMAD3 (Ser423/425), anti-p27Kip1, anti-SOX2 (Cell Signaling Technology, Beverly MA, USA), anti-OLIG2, anti-β-Tubulin beta III isoform (Millipore, Temecula, CA, USA), anti-CYCLIN B1, p21CIP1 (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-GFAP (BD Pharmingen San Jose, CA), anti-NESTIN (R&D Systems, Minneapolis, MN, USA), and anti-CYCLIN D1 (ThermoFisher Scientific, Waltham, MA USA).
Anti nestin
Manufactured by R&D Systems
Sourced in United States
Anti-Nestin is a protein that functions as an intermediate filament. It is commonly used as a marker for neural stem and progenitor cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti oct4, by Santa Cruz Biotechnology (4 mentions)
Anti map2, by Merck Group (3 mentions)
Anti sox2, by Merck Group (3 mentions)
Anti afp, by Agilent Technologies (3 mentions)
Anti nanog, by R&D Systems (3 mentions)
Anti ssea 4, by Developmental Studies Hybridoma Bank (3 mentions)
Tuj1 anti tubulin beta 3 isoform, by Merck Group (3 mentions)
Anti musashi, by Merck Group (3 mentions)
Anti sma, by Agilent Technologies (3 mentions)
Anti lc3b, by Cell Signaling Technology (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
At8 anti p tau, by Thermo Fisher Scientific (2 mentions)
Anti tra 1 81, by Merck Group (2 mentions)
Anti tbr1, by Abcam (2 mentions)
Anti ctip2, by Abcam (2 mentions)
Normal horse serum, by Vector Laboratories (2 mentions)
Ag1478, by Merck Group (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti β actin peroxidase conjugated antibody, by Merck Group (1 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (1 mentions)
13 protocols using anti nestin
1
Molecular Mechanisms of Glioblastoma Response to Temozolomide
2
Immunocytochemical Characterization of Human NPCs
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Human NPCs were characterized by immunocytochemistry. Briefly, cells grown on glass coverslips were fixed with 4% paraformaldehyde and incubated in the blocking buffer (5% goat serum, 1% bovine serum albumin, and 0.1% Triton X-100) for 15 min. Cells were then incubated in primary antibodies diluted in the blocking buffer at 4 °C overnight. Appropriate secondary antibodies were used for single and double labeling. All secondary antibodies were tested for cross-reactivity and nonspecific immunoreactivity. The following primary antibodies were used, anti-SOX2 (1:200, R&D Systems), anti-Nestin (1:500, R&D Systems), anti-SOX1 (1:250, Millipore). Bis-benzamide (DAPI, 1:1000; Sigma) was used to visualize the nuclei. Images were captured using a Zeiss Axiovision microscope with z-stack split view function.
3
Antibody Validation and ER Stress Assays
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Anti-V5, FLAG and GAPDH antibodies were purchased from Proteintech. Anti-α-Tubulin and anti-HA antibodies were purchased from Sigma. Anti-Sox2, Nanog and Oct4 antibodies were purchased from Abcam. The following antibodies were used: anti-FKBP9 (Invitrogen), anti-Calnexin (Santa Cruz), anti-Nestin (R&D Systems), anti-pmTOR (Invitrogen), anti-pP70S6K (Millipore), anti-pERK1/2 (Promega), anti-p65 (EPITOMICS), anti-ASK1 (Santa Cruz), anti-pASK1 (Santa Cruz), anti-pIRE1α (NOVUS). Other antibodies for immunoblotting were purchased from Cell Signaling Technology. Aggresome Detection Kit was purchased from Abcam. Thapsigargin (Tg) and tunicamycin (Tm) were purchased from Apexbio. Proteasome inhibitor MG132 and lysosomal inhibitors Bafalomycin A1 (Baf A1) and chloroquine (CQ) were obtained from Sigma. Drugs were dissolved and stored at − 20 °C or − 80 °C according to the instructions.
4
Spinal Cord Injury Nestin Expression
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At 1 week after SCI, six rats from each of the three groups were deeply anesthetized by an intraperitoneal injection of ketamine and were intracardially perfused with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PB; pH=7.4). The thoracic spinal cord was excised, postfixed for 24 hours, and maintained overnight in 30% sucrose in 0.1 M phosphate buffer solution. Spinal cord tissues were sectioned at a thickness of 30 μm on a cryostat, and sections were floated on the surface of 0.1 M phosphate buffer solution. A 5 and 6 mm rostral to the center of injury was performed. To detect nestin (a marker for NSPCs), spinal cord sections were blocked with 4% normal serum in 0.5% Triton X-100 for 1 hour at room temperature, incubated overnight at 4°C with a 1 : 200 dilution of mouse monoclonal anti-nestin (R&D Systems, Inc., Minneapolis, MN, USA) and rabbit polyclonal anti-BrdU (Abcam, Cambridge, MA, USA), and then rinsed for three times for 10 minutes in a 0.1 M PB. Sections were incubated for 2 hours with Alexa Fluor 594 goat anti–mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG (both 1 : 300; invitrogen). The images were viewed on a computer monitor using a Zeiss Plan-Apochromat 40X objective (Carl Zeiss Meditec Inc., Jena, Germany). Enumeration of immune-positive cells used a Labworks, version 4.5, computer-assisted image analyzer (UVP, Upland, CA, USA).
5
Immunostaining of Pluripotent and Differentiated Cells
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iPSCs or differentiation culture were fixed with 4% paraformaldehyde in PBS. After permeablization in 1% triton X-100/PBS for 20 min, immunostaining were performed using the following primary antibodies: anti-Oct4 (Santa Cruz), anti-Nanog (Santa Cruz), anti-SSEA1 (Millipore), anti-Gata4 (Santa Cruz), anti-SMA (Abcam), anti-Nestin (R&D). Secondary antibodies used were Alexa Fluor 488/546 anti-mouse IgM, and Alexa Fluor 488/546 anti-mouse or anti-rabbit IgG (Invitrogen). DAPI (Vector Laboratories) was used for staining the nuclei.
6
Immunocytochemical and Western Blot Analysis
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Immunocytochemistry and Western blot analysis were performed as we described before.27 , 28 The following primary antibodies were used: anti‐OCT4 (1:200; Santa Cruz), anti‐SOX2 (1:200; Millipore), anti‐NANOG (1:200; R&D Systems), anti‐SSEA‐4 (1:100; Developmental Studies Hybridoma Bank), anti‐TRA‐1‐81 (1:100; Chemicon), Tuj1 anti‐tubulin beta III isoform (1:200; Millipore), anti‐SMA (1:100; DAKO); anti‐AFP (1:100; DAKO), anti‐Nestin (1:200; R&D Systems), anti‐Musashi (1:200; Millipore), anti‐Map2 (1:200; Millipore), anti‐TBR1 (1:100; Abcam), anti‐CTIP2 (1:100; Abcam), Aβ42 anti‐β‐Amyloid42 (1:500; Calbiochem), AT8 anti‐p‐Tau (1:1000; Thermo Fisher Scientific) and anti‐LC3B (1:500; Cell Signaling), Tau5 anti‐tau (1:1000; Thermo Fisher Scientific), anti‐Mfn1 (1:1000; Abcam), anti‐Mfn2 (1:1000; Cell Signaling), anti‐DRP1 (1:1000; Cell Signaling), anti‐Fis1 (1:1000; Santa Cruz), anti‐Ub (1:4000; Santa Cruz), anti‐LAMP2 (1:1000; Santa Cruz), anti‐Beclin1 (1:1000; Cell Signaling), p62 anti‐SQSTM1 (1:1000; Santa Cruz) and anti‐β‐actin (1:10 000; Santa Cruz).
7
Immunostaining of Pluripotent Stem Cells
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Cells were cultured on coverslips were fixed and then probed with primary antibody, washed with PBST, probed with secondary antibody in the dark and washed with PBST again. The cells were stained with Vectashield mounting medium containing 4′, 6- diamidino-2-phenylindole (DAPI; Vector Laboratories) before observed under a confocal microscope (Olympus FV1000). Primary antibodies used for probing iPS cells were anti-Oct4 (sc-8628, Santa Cruz), anti-SSEA-1 (mab34301, Millipore) and anti-Nanog (sc-33760, Santa Cruz). As for probing EBs, primary antibodies used were anti-alpha smooth muscle Actin (ab5694, Abcam), anti-Gata4 (sc-25310, Santa Cruz) and anti-Nestin (mab2736, R&D). Secondary antibodies used to probe iPS cells were Alexa Fluor 594 lgG antibody (Invitrogen) while Alexa Fluor 488 lgG antibody (Invitrogen) was used to probe EBs.
8
Immunofluorescence Staining of Neural Markers
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Samples were fixed with 4% paraformaldehyde (Biosesang) overnight at 4°C. The fixed samples were permeabilized and washed with TPBS containing 0.2% Triton X‐100 (Sigma) and 0.01% Tween‐20 (Biosesang) in PBS blocked with 3% normal horse serum (Vector Labs) in TPBS for 2 h at RT. The following primary antibodies were prepared in blocking solution for incubation overnight with gentle rocking at 4°C: anti‐Nestin (1:200; R&D Systems), anti‐MAP2 (1:200; Thermo Fisher Scientific), anti‐S100β (1:200; Abcam), anti‐GFAP (1:200; DAKO), anti‐β‐amyloid (anti‐Aβ), 1–16 antibody (clone 6E10) (1:100; Biolegend), anti‐LC3B (1:100; Cell Signaling), and anti‐human mitochondria (anti‐hMito) (1:100; Abcam). Afterward, the cells were washed with TPBS three times and were incubated with secondary antibodies (Thermo Fisher Scientific) for 2 h at RT with gentle rocking, and then counterstained with DAPI for 30 min in PBS after additional three washes with TPBS. The fluorescence images were acquired using the ImageXpress Micro Confocal (IXMC) microscopy for high content analysis with the Metaxpress6 program v.6.6.3.55 (Molecular Devices).
9
Differentiation of hHF-MSC-derived iPSCs
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hHF-MSC-derived iPSCs were harvested by collagenase type IV. After settling, the supernatant was aspirated and the MEF medium was replaced to remove the MEF. hHF-MSC-derived iPSCs were transferred to petri dishes in the MEF medium. After an 8 days floating culture, embryoid bodies were transferred to gelatin-coated plates and were then incubated for another 16 days. After the incubation, the cells were fixed with 4% paraformaldehyde in PBS and then incubated in PBS containing 5% normal goat serum (Maixin Biotech, Fuzhou, China), 1% bovine serum albumin (BSA, Biotopped, China), and 0.2% Triton X-100. The primary antibodies were as follows: anti-alpha smooth muscle actin (α-SMA, R&D, USA), anti-alpha fetoprotein polyclonal antibody (AFP, R&D, USA), and anti-Nestin (R&D, USA). vimentin(Cell Signaling, USA), desmin (Cell Signaling, USA), βIII-tubulin (Cell Signaling, USA). The secondary antibodies were Alexa 555-labeled anti-mouse IgG (1:1000, Cell Signaling, USA). Nuclei were stained with 1 mg/ml Hoechst 33342 (Invitrogen, USA).
10
Immunophenotyping of Neural Stem Cells
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The primary antibodies used were: anti-filaggrin (1:500; Abcam, catalog number # ab24584), anti-fibronectin (1:200; R&D Systems, catalog number # AF19181), anti-GFAP (1:400; R&D Systems, catalog number # AF2594), anti-βIII tubulin (1:200; R&D Systems, catalog number # MAB1195), anti-vimentin (1:200; R&D Systems, catalog number # MAB2105), anti-nestin (1:200; R&D Systems, catalog number # MAB2736), anti-CNPase (1:400; Chemicon, catalog number # MAB326), CD34-FITC (Pharmingen, San Jose, CA catalog number # 553733) and anti-p63 (1:500, AbCAM, catalog number # ab53039). The secondary antibodies were: Texas Red anti-sheep (1:1,000 catalog number # TI-6000), FITC anti-sheep (1:500 catalog number # F7634), FITC anti-rabbit (1:1,000 catalog number # FI-1000), anti-sheep HRP (1:1,000 catalog number # A16-147), anti-rabbit HRP (1:1,000 catalog number # PI-1000) all from Vector Laboratories, USA. The fluorophore-conjugated antibodies for flow cytometry were: CD34-PE catalog number # 551387, CD31-APC catalog number #561814, CD45-PE catalog number # 553081, CD44-PE catalog number # 561860, CD90-FITC catalog number # 554894, CD29-PE catalog number #562801, CD105-PE catalog number # 562759 diluted 1:500, all from BD Biosciences (San Jose, CA, USA).
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