The hippocampus was imaged by magnetic resonance imaging (NiuMai, Suzhou, China). An electron microscope (Thermo, Waltham, MA, USA) was used to image neurovascular units of the hippocampus. The hippocampal neurons of normal rats were extracted by separation of hippocampal tissue with scissors and Hank’s buffer solution (Gibco, Carlsbad, CA, USA), and digestion with trypsin (Gibco, Carlsbad, CA, USA), and implantation or culture with DMEM/F-12 medium (Gibco, Carlsbad, California, USA) attached 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1% 10 000 units/ml penicillin-10000 μg/ml streptomycin (Gibco, Carlsbad, California, USA). Cells were seeded in a 75-mm dish (Corning, Corning, USA) for 24 h and then washed twice with phosphate-buffered saline (PBS; Gibco, Carlsbad, CA, USA). Then, cells were incubated at room temperature for 2 h with NSE antibody (ab53025, Abcam, Cambridge, UK) mixed with TBST, and the dilution of ratio was performed according to the manufacturer’s instruction. Then, the goat anti-rabbit antibody (ab150077, Cambridge, UK) incubated cells for 2 h after the cells were washed for 3 times with TBST. ECL kit (Invitrogen, Carlsbad, California, USA) added to cell, then the cells were washed with PBS. DAPI mixed with PBS stained the cells for 30 min. The fluorescence of the cells was observed by fluorescence microscope (Olympus, Tokyo, Japan).
Ab53025
Manufactured by Abcam
Sourced in United Kingdom
Ab53025 is a laboratory equipment product. It is a device used for scientific research and analysis purposes. The core function of this product is to facilitate specific experimental or measurement tasks in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Ab18207, by Abcam (2 mentions)
Electron microscope, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Ecl kit, by Thermo Fisher Scientific (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Ab227978, by Abcam (1 mentions)
Vectastain elite abc rabbit immunoglobulin g kit, by Vector Laboratories (1 mentions)
Ab96792, by Abcam (1 mentions)
Ab14849, by Abcam (1 mentions)
Ab104225, by Abcam (1 mentions)
Peroxidase conjugated goat anti mouse igg, by Proteintech (1 mentions)
Ab1761, by Merck Group (1 mentions)
S9442, by Merck Group (1 mentions)
Sc 166574, by Santa Cruz Biotechnology (1 mentions)
Peroxidase conjugated goat anti rabbit igg, by Proteintech (1 mentions)
Ab3329, by Abcam (1 mentions)
10 protocols using ab53025
1
Imaging Hippocampal Neurons and Vasculature
2
Immunohistochemical Staining Protocol
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The anti–ENO1 rabbit antibody (Anit‐ENO1 #ab227978, Abcam), anti–ENO2 rabbit antibody (Anti–NSE, #ab53025, Abcam), anti–BRAF V600E rabbit antibody (Anti–BRAF mutated V600E #200535, Abcam), and the VECTASTAIN Elite ABC Rabbit Immunoglobulin G Kit (Vector Laboratories) were used for immunohistochemical staining. The slides with antibody were diluted 1:2000 (Anti–ENO1), 1:200 (Anti–BRAFV600E) and 1:100 (Anti–NSE) were incubated overnight at 4°C. We assigned an intensity score of +2 to cytoplasm that was stained as intensely as the positive control, a score of +1 when the cytoplasmic staining was weaker than the positive control, and a score of 0 to unstained cytoplasm.
3
Antibody Staining Protocol for Kidney Tissue
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Antibodies were purchased as follows: Primary antibodies: rabbit-anti-UCH-L1 (AB1761,MILLIPORE), rabbit-anti-synaptopodin (S9442, Sigma-Aldrich), mouse-anti-α-actinin4 (MAB1682, MILLIPORE), mouse-anti-nestin (MAB2763, R&D), rabbit-anti-NeuN (ab104225, Abcam), mouse-anti-S100 (ab14849, Abcam), rabbit-anti-NSE (ab53025, Abcam), mouse-anti-nephrin (sc-166574, Santa Cruz Biotechnology), mouse-anti-WT-1 (ab96792, Abcam). Secondary antibodies: peroxidase-conjugated goat-anti-mouse IgG, peroxidase-conjugated goat-anti-rabbit IgG, fluorescein (FITC)-conjugated goat-anti-rabbit/mouse IgG, cy3-conjugated goat-anti-mouse/rabbit IgG all from Protein Tech Group Inc., Chicago, USA, HRP-goat-anti-mouse IgG, HRP-goat-anti-rabbit IgG from Long Island Biotech Co., LTD, Shanghai, China.
4
Proteomic Profiling of Mouse Brain Samples
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Mouse brain samples were extracted using the same method described above for the MS analysis. For each analysis, 5–20 μg of sample was added per lane, separated on 12% Bis-Tris Pre-Cast gels (Thermo Fisher) and transferred to PVDF membranes using 7.5% BSA in TBS for blocking. All primary antibodies were used overnight at 1:1000. Antibodies were used against TH (tyrosine hydroxylase; EMD Millipore, 657012 RRID:AB_696697 ), DAT (dopamine transporter; EMD Millipore, MAB369 RRID:AB_2190413 ), Ddc (aromatic-L-amino-acid decarboxylase; Abcam, ab3905 RRID:AB_304145 ), NSE (neuron specific enolase; Abcam, ab53025 RRID:AB_881756 ), PKC-β2 (protein kinase C beta-2; Abcam, ab32026 RRID:AB_779042 ), Akt (protein kinase B; Cell Signaling, 9272 RRID:AB_329827 ), Lmp7 (proteasome subunit beta type-8; Abcam, ab3329 RRID:AB_303708 ), and α-synuclein clone D37A6 (Cell Signaling Technology, #4179 RRID:AB_1904156 ) or clone Syn211 (Sigma-Aldrich Cat# S5566, RRID:AB_261518 ). Antigen-antibody complexes were detected using an Odyssey LC scanner (LiCor) after incubation with appropriate secondary antibodies. Densitometry was used to quantify intensity of protein bands.
5
Immunofluorescent Analysis of Neurogenic Transdifferentiation
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To assess neurogenic transdifferentiation, the cells were stained with neuron-specific enolase (NSE), Tuj-1 (Neuron-specific class III beta-tubulin) and glial fibrillary acidic protein (GFAP). Briefly, cells were fixed with 4% paraformaldehyde for 20 min. Then the cells were permeabilized using PBS containing 0.5% Triton-X 100 (Sigma Aldrich) for 1 hour and then blocked using PBS supplemented with 4% BSA and 0.3% Triton-X 100 for another hour. After these treatments, the cells were incubated primary anti-NSE antibody (1:500, ab53025, Abcam, Cambridge, MA, USA), anti-Tuj-1 (1:500, ab18207, Abcam) or anti-GFAP (1:200, ab48050, Abcam) overnight at 4°C. Immunolabeling was visualized using DyLight® 594-conjugated Donkey anti-rabbit IgG polyclonal antibody (1: 1000, Ab96921, Abcam) for 2 h at room temperature in PBS. The nuclei were stained with 5 mg/ml DAPI for 15min and were examined on a fluorescent microscope. Digital Immunofluorescent images were obtained using an Olympus IX71 inverted microscope (Olympus, Tokyo, Japan).
6
Quantification of Newly Synthesized Proteins
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Following BONCAT purification, equal volumes of the elution fraction were loaded and separate by SDS-PAGE and analysed via western blotting as previously described (Evans et al., 2019 (link)). The total amount of newly synthesised proteins was quantified using the REVERT total protein stain (LI-COR, 926–11010), with representative proteins for each cluster were detected using the following primary antibodies: α adaptin (ThermoFisher, MA3-061, 1:500), neuron specific enolase (NSE) (Abcam, ab53025, 1:500), AT6V1B2 (Abcam, ab73404, 1:500), PP2A-A (Sigma-Aldrich, 07–250, 1:1000), AUF-1 (Sigma-Aldrich, 07–260, 1:500), and GAPDH (Millipore, MAB374, 1:1000).
7
Apoptosis and Glial Activation in HIBD
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TUNEL assay and immunofluorescence double-labeling staining of TLR2 with NSE, GFAP or Iba1 were performed on the brains of rats in the control, HIBD and MSCs groups. Briefly, all rats were anesthetized and transcardially perfused with 4 % paraformaldehyde (Kelong Chemical Co. Ltd, China). The 12-μm slices of brain tissue were sectioned using a Leica CM3050 S cryostat. Apoptosis was detected using a TUNEL kit (KeyGEN BioTECH, China) following the manufacturer’s protocol. The number of TUNEL-positive cells was counted from four randomly selected fields in each group, and the counts were expressed as a percentage of the total number of cells.
Double stainings for TLR2 and NSE, GFAP or Iba1 were performed to evaluate their colocalization by using the following antibodies: goat anti-TLR2 (1:50, Santa Cruz, sc-16237) together with rabbit anti-NSE (1:100, Abcam, ab53025) or mouse anti-GFAP (1:100, Abcam, ab10062) or rabbit anti-Iba1 (1:50, Santa Cruz, sc-32725) overnight at 4 °C. The primary antibodies were visualized using Alexa Fluor 488-conjugated chicken anti-rabbit (anti-mouse) IgG (1:100, Life Technologies) together with Alexa Fluor 594-conjugated donkey anti-goat IgG (1:100, Jackson), and 4’,6-diamidino-2-phenylindole (DAPI, 1:100, Yeasen) was used to stain the nucleus. The images were captured using a Nikon laser confocal microscope (Nikon, Japan).
Double stainings for TLR2 and NSE, GFAP or Iba1 were performed to evaluate their colocalization by using the following antibodies: goat anti-TLR2 (1:50, Santa Cruz, sc-16237) together with rabbit anti-NSE (1:100, Abcam, ab53025) or mouse anti-GFAP (1:100, Abcam, ab10062) or rabbit anti-Iba1 (1:50, Santa Cruz, sc-32725) overnight at 4 °C. The primary antibodies were visualized using Alexa Fluor 488-conjugated chicken anti-rabbit (anti-mouse) IgG (1:100, Life Technologies) together with Alexa Fluor 594-conjugated donkey anti-goat IgG (1:100, Jackson), and 4’,6-diamidino-2-phenylindole (DAPI, 1:100, Yeasen) was used to stain the nucleus. The images were captured using a Nikon laser confocal microscope (Nikon, Japan).
8
Neurogenic Induction of gADSCs and gMDSCs
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For neurogenic induction, 70% confluent gADSCs and gMDSCs were cultured in DMEM/F12 medium supplemented with 10 ng/mL epidermal growth factor (G5021; Promega, Madison, WI) plus 10 ng/mL basic fibroblast growth factor (bFGF) (G5071, Promega, Madison, WI) for 24 h. Subsequently, the medium was changed to DMEM/F12 supplemented with 1 mM β-mercaptoethanol (21985-023, Gibco, Gaithersburg, MD) and 10% FBS for 6 h for induction. The cells were then washed three times with PBS to remove β-mercaptoethanol and cultured in DMEM/F12 supplemented with 2% DMSO, 200 μM butylated hydroxyanisole (20021; Sigma–Aldrich, St. Louis, MO) and 20 ng/mL bFGF. When neuron-like cells were observed, the cells were stained with neuron-specific enolase (NSE) antibody (ab53025; Abcam, Cambridge, UK) and examined by immunofluorescence [11] (link).
9
Quantifying Neurogenic Markers in ADMSCs
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On day 1 after the second step of induction, the ADMSCs were subjected to flow cytometry to quantify the proportion of cells with positive neurogenic markers. The cells were fixed using 4% paraformaldehyde at room temperature for 30 min and permeabilized using 0.1% TritonX-100+2% BSA in PBS at 37°C for another 30 min. Then the cells were incubated with primary anti-NSE antibody (1:100, ab53025, Abcam) for one hour, anti-GFAP (1:100, ab10062, Abcam) for 30 min or anti-Tuj-1 (1:100, ab18207, Abcam) for 30 min. For anti NSE and anti-Tuj-1, the secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (1:4000, ab150077, Abcam) for 30 min at 22°C. For anti-GFAP, the secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (1:500, ab96879, Abcam) for 30 min at 22°C. The proportion of cells with active neuronal markers were then analyzed using a FACSCaliber (BD Biosciences, Franklin Lakes, NJ, USA). Data acquisition was done using CellQuest 3.2 software (BD Biosciences). Each test was performed with at least three repeats.
10
Immunohistochemical Analysis of NSE Expression
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NSE expression in the tumor-bearing tissues from nude mice and in the primary NB tissues from the pediatric patient were analyzed by immunohistochemical staining with anti-NSE antibody (ab53025, Abcam, UK) at 1:100 dilution. Positive NSE protein expression appeared as a dense brown color and was mainly located on the cell membrane; occasionally, positive protein expression was found in the cytoplasm.
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