For immunofluorescence of mouse tissue, tumor bearing mice were cardially perfused with PBS, followed by 4.5% phosphate-buffered formaldehyde solution (Roti-Histofix 4.5%, 2213, Carl Roth). Brain tissue was incubated in 30% sucrose solution overnight for cryoprotection and snap frozen afterwards. Heat-induced epitope retrieval with 0.01 M citrate buffer, pH 6.0, was performed. The following primary antibodies were used: anti-nestin (1:400, ab6320, Abcam, RRID:AB_308832), anti-CD31 (1:100, AF3628, R&D Systems, RRID:AB_2161028), anti-aquaporin 4 (1:200, ab9512, Abcam, RRID:AB_307299), anti-activated Notch1 (1:50, ab8925, Abcam, RRID:AB_306863) and anti-ki67 (1:500, ab15580, Abcam, RRID:AB_443209). Donkey anti-mouse IgG Alexa Fluor 488 (1:400; A-21202, Thermo Fisher Scientific, RRID:AB_141607), goat anti-mouse IgG Alexa Fluor 488 conjugate (1:400; A-11029, Thermo Fisher Scientific, RRID:AB_138404), donkey anti-goat IgG Alexa Fluor 633 (1:400; A-21082, Thermo Fisher Scientific, RRID:AB_141493) and donkey anti-rabbit IgG Alexa Fluor 546 (1:400; A-10040, Thermo Fisher Scientific, RRID:AB_2534016) secondary antibodies were used. Images were acquired using a Leica TCS SP5 confocal microscope.
Ab6320
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab6320 is a primary antibody produced by Abcam. It is designed for use in immunohistochemistry and western blotting applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab15580, by Abcam (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Tcs sp5 confocal microscope, by Leica (2 mentions)
Roti histofix 4.5, by Carl Roth (2 mentions)
Ab19898, by Abcam (2 mentions)
Ab7260, by Abcam (2 mentions)
Lab tek chamber, by Thermo Fisher Scientific (2 mentions)
Pab12663, by Abcam (2 mentions)
Myelin basic protein, by Abcam (2 mentions)
βiii tubulin, by Merck Group (2 mentions)
Bt 575, by Biomedical Technologies (2 mentions)
A 21082, by Thermo Fisher Scientific (1 mentions)
A21202, by Thermo Fisher Scientific (1 mentions)
A11029, by Thermo Fisher Scientific (1 mentions)
Ab9512, by Abcam (1 mentions)
Af3628, by R&D Systems (1 mentions)
A10040, by Thermo Fisher Scientific (1 mentions)
Ab8925, by Abcam (1 mentions)
Prolong gold with dapi, by Thermo Fisher Scientific (1 mentions)
Ab28365, by Abcam (1 mentions)
16 protocols using ab6320
1
Immunofluorescence Analysis of Mouse Brain Tissue
2
Immunostaining and in situ hybridization protocol
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Immunostaining was performed using 8-μm cryosections as previously described.16 (link) Antibodies in this study were anti-ETV2 (N-term) (AP11311a, ABGENT), anti-ETV2 (C-term) (AB65825, Abcam), anti-ETV2(Internal)(LS-C61735, Isbio), anti-hCD31 (AB32457, Abcam), anti-CD31 (AB9498, AB28365, Abcam), anti-ki67 (AB156956, AB15580, Abcam), anti-CD34 (AB157304, Abcam), anti-VE-cad (AB7047, Abcam), anti-flk1 (#2478, CST), anti-nestin (ab6320, Abcam), anti-CD133 (AB19898, Abcam), anti-GFAP (AB7260, Abcam), anti-NeuN (SAB4300883, Sigma), and anti-Sox2 (AB97959, Abcam). Sections were mounted using Prolong Gold with DAPI (Life Technologies). In situ hybridization was performed as previously described.16 (link)
3
Immunocytochemical Characterization of Differentiated hMSCs
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The assay was performed as previously described7 (link). Briefly, fixed cells were incubated overnight at 4 °C with primary monoclonal antibodies against Nestin (1:400, #ab6320), MAP2 (1:250, #ab32454), TH (1:200, #ab112) and DAT (1:200, # ab92868) (Abcam, USA). After the induction period, hMSCs were washed five times with PBS and incubated with AF488 and AF594 conjugated secondary antibodies (1:500, Abcam, USA) for 1 h at room temperature (RT). Finally, after washing five times with PBS, cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (#10236276001, Sigma, USA) to visualize the cell nuclei. Cells were washed thrice with PBS to remove excess DAPI stain. Stained cells were examined using a fluorescence microscope equipped with a digital camera (Nikon Eclipse 80i, Japan).
4
Nestin Expression in CSC1589 Cells
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CSC1589 cells were cultured in 12-well plates at density of 1×105 cells/well and treated with JQ1 for 0.125 or 0.25 µM. Single cell suspensions were seeded on coverslips pre-coated with fibronectin (1 µg/ml, cat. no. F1141-5MG, Sigma-Aldrich; Merck KGaA) and ornithine (50 mg/ml, P3655-50MG, Sigma-Aldrich; Merck KGaA) overnight. CSC1589 cells were fixed with 4% paraformaldehyde at room temperature for 10-15 min and permeabilized with 0.3% Triton X-100 (Sigma-Aldrich; Merck KGaA) in PBS for 5 min. Then, potential non-specific binding sites were blocked with antibody dilution buffer [10% fetal bovine serum and (Sigma-Aldrich; Merck KGaA) and 1% bovine serum albumin (Gentihold) in PBS]. Sections was incubated with an antibody against Nestin (ab6320, 1:50; Abcam) overnight at 4°C. After washing the sections with PBS for 5 min, secondary antibodies were applied [Donkey anti-Mouse IgG (H+L) ReadyProbes™ Secondary Antibody, Alexa Fluor® 488, R37114, 1:5,000; Invitrogen; Thermo Fisher Scientific, Inc.] for 30 min at room temperature before mounting in water soluble mounting medium (Wuhan Boster Biological Technology, Ltd.) with DAPI (room temperature, 2 min). After the final washing step by PBS, the glass coverslips were mounted upside-down and visualized with an Olympus BX53 microscope at ×400 magnification.
5
Proliferation and Differentiation of hAMSCs
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Ki67 staining was used to detect the proliferation capacity of hAMSC in response
to GDNF. These stem cells were seeded on 24-well plates at 1 × 104 in
MSC complete media with or without GDNF (100 ng/ml). After 3 days, the cells
were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 45 min,
following which they were pre-incubated in PBS containing 0.3% Triton X-100 with
10% goat serum prior to incubation with Ki67 antibody (1:500; ab15580, Abcam,
Cambridge, MA, USA). Alexa Fluor-labeled secondary antibody was added to the
sections and incubated for 2 h (1:500; Invitrogen, Carlsbad, CA, USA), and
4′,6-diamidino-2-phenylindole (DAPI) was incubated for 0.5 h (Invitrogen) to
visualize markers and cell nuclei. To determine the differentiation capacity of
hAMSCs, we performed immunostaining for Nestin (1:500; ab6320, Abcam), glial
fibrillary acidic protein (GFAP; 1:500, ab7260, Abcam), and Tuj-1 (1:500;
PA5-85874, ThermoFisher), as described above. The number of positive cells was
visualized and recorded using an inverted fluorescence microscope (Zeiss,
Oberkochen, Germany). All measurements were performed according to the
recommended protocol.
to GDNF. These stem cells were seeded on 24-well plates at 1 × 104 in
MSC complete media with or without GDNF (100 ng/ml). After 3 days, the cells
were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 45 min,
following which they were pre-incubated in PBS containing 0.3% Triton X-100 with
10% goat serum prior to incubation with Ki67 antibody (1:500; ab15580, Abcam,
Cambridge, MA, USA). Alexa Fluor-labeled secondary antibody was added to the
sections and incubated for 2 h (1:500; Invitrogen, Carlsbad, CA, USA), and
4′,6-diamidino-2-phenylindole (DAPI) was incubated for 0.5 h (Invitrogen) to
visualize markers and cell nuclei. To determine the differentiation capacity of
hAMSCs, we performed immunostaining for Nestin (1:500; ab6320, Abcam), glial
fibrillary acidic protein (GFAP; 1:500, ab7260, Abcam), and Tuj-1 (1:500;
PA5-85874, ThermoFisher), as described above. The number of positive cells was
visualized and recorded using an inverted fluorescence microscope (Zeiss,
Oberkochen, Germany). All measurements were performed according to the
recommended protocol.
6
Immunocytochemical analysis of neural markers
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Immunocytochemical analysis was performed as previously described. Briefly, the cultured cells or neurospheres were fixed in 4% paraformaldehyde-PBS for 30 min, blocked in PBS containing 5% goat serum and 0.3% TritonX-100 at 37 °C for 1 h, and then incubated with one of the following primary antibodies overnight at 4 °C: rabbit anti-GS (1:100, Abcam, ab73593), mouse anti-vimentin (1:75, Abcam, ab8976), rabbit anti-Sox2 (1:100, Abcam, ab92494), mouse anti-nestin (1:100, Abcam, ab6320), mouse anti-rhodopsin (1:50, Abcam, ab5417). Following PBS wash, cells or neurospheres were incubated in fluorophore-conjugated goat anti-mouse IgG (ZSGB-BIO, China) or goat anti-rabbit IgG (Multi Sciences, China) for 2 hours in the dark and then counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma, USA) for five minutes. Fluorescent images were recorded using confocal microscopy (Leica SP8, Germany) or fluorescent microscopy (Leica DM5000B, Germany).
7
Antibody-Based Stem Cell Analysis
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8
Characterization of U87 Spheroid and Neural Stem Cell Models
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Spheroids of U87 cells were grown for three days using the hanging drop method. Each droplet contained 20,000 cells in 20 μL medium. Each spheroid was plated into a well of a 96-well plate containing 50 μL of 2% agarose in PBS after waiting 5 min for the gel to solidify. The wells were filled with 100 μL media. After 24 h, the spheroids became smaller and tightly packed. Neurospheres of CSCs were prepared by culturing in NSC media, as described above. Spheroids were cryosectioned in 20 μm slices, stained, and then imaged by confocal microscopy in Lab-tek chambers (Thermo Fisher). Cells were stained with antibodies for Nestin (ab6320) (Abcam) and CD133 (PAB12663) (Abcam) for neural stem cell markers. Antibodies to glial fibrillary acidic protein (BT-575) (Biomedical Technologies Inc., Mt. Arlington, NJ), βIII-tubulin (Sigma-Aldrich), and myelin basic protein (Abcam) were used as markers of differentiated cells. DAPI (Invitrogen) was the nuclear stain used.
9
EdU Labeling and Immunostaining in Tissue
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Animals were treated with 50 mg/kg EdU i.p. (BCK488-IV-IM-S, baseclick GmbH) 4 h prior to euthanasia. Animals were cardially perfused with 4.5% phosphate-buffered formaldehyde solution (Roti-Histofix 4.5%, 2213, Carl Roth). Tissue was frozen after cryoprotection with 30% sucrose solution and 10 µm cryosections were cut. The sections were permeabilized with 0.5% Triton X-100 in PBS for 20 min. The EdU Click Kit (BCK488-IV-IM-S, baseclick GmbH) was used for EdU detection. After washing, sections were incubated with the reaction cocktail for 30 min as per the manufacturer’s instructions. Afterwards sections were incubated with anti-nestin (1:400, ab6320, Abcam, RRID:AB_308832) and anti-CD31 (1:50; PA5-16301, ThermoFisher Scientific, RRID:AB_10981955) antibodies.Goat anti-rabbit Alexa Fluor 594 (1:400; A-11037, ThermoFisher Scientific, RRID:AB_2534095) and goat anti-mouse Alexa Fluor 633 (1:400; A-21052, ThermoFisher Scientific, RRID:AB_2535719) secondary antibodies were used. Images were acquired using a Leica TCS SP5 confocal microscope (LAS software version 2.7.3.9723).
10
Irradiation and Pharmacological Modulation of Stemness
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Cells were first irradiated with 4 Gy and then treated with 10 μM RO4929097, 10 μM TMZ as well as their combination. 4 days post-treatment total cell lysate was prepared using SDS lysis buffer. Lysates were blotted onto a PVDF-membrane. Membranes were probed overnight at 4°C with primary antibodies and bound antibodies were visualized using HRP-linked secondary antibodies (Cell-Signaling) and ECL Luminescence (Pierce Biotechnology). Anti-SOX2 (ab75485, Abcam), anti-Nestin (ab6320, Abcam), anti-beta-tubulin III (T3952, Sigma-Aldrich), and anti-Lamin A/C (Sigma-Aldrich) were used at 1:1000 dilution.
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