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6 protocols using cf488a donkey anti mouse igg

1

Differentiation of P19 Cells into Neurons

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P19 cells were grown in MEM alpha medium containing 7.5% bovine calf serum, 2.5% fetal bovine serum, with penicillin and streptomycin on 10cm tissue culture plates. To induce differentiation, cells were subjected to a previously described [23 (link)] protocol with minor modification. Briefly, cells were cultured for 48 h in the presence of 1μM ATRA and then dispersed with 0.25% trypsin/EDTA. The P19 cells were then seeded into fresh ATRA-containing medium and additional 24 h. Cells formed small aggregates from which individual aggregates were seeded into 6-well plates containing 20mm2 gelatin-coated coverslips in ATRA-containing medium. Incubation was at 37°C, 5% CO2 and medium was changed every two days. On day 8 (day 11 of ATRA treatment), cells were fixed and permeabilized with 4% paraformaldehyde, then stained for EphrinB1 (Santa Cruz) with secondary CF488A donkey anti-mouse IgG (Biotium), and counter-stained for DAPI (Vector Laboratories, Inc.). Immunostained cells were visualized using the Leica AF6000 Modular System fitted with a Leica CTR 6000 light/laser source and Leica DMI 6000B microscope running the Leica Application Suite Advanced Fluorescence Software. Cells were photographed under 20X magnification.
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2

Platonin Neuroprotection Protocol

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Platonin was synthesized by and obtained from Gwo Chyang Pharmaceuticals (Tainan, Taiwan; Figure 1A). Primary antibody against cleaved-caspase-3 was purchased from Abcam (Cambridge, UK). The anti-Iba1 antibody was purchased from Wako Chemicals USA (Richmond, VA, USA). The anti-NeuN antibody was from Merck Millipore (Darmstadt, Germany). For immunostaining, CF488A donkey anti-mouse IgG and CF488A donkey anti-rabbit IgG were purchased from Biotium (Fremont, CA, USA). Dihydroethidium (DHE) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Platonin was dissolved in phosphate-buffered saline (PBS) and stored at 4 °C.
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3

Immunostaining and Cell Proliferation Assays

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Immunohistochemistry was performed as follows: fixation with 4% PFA for 20 min at room temperature; washing twice in PBS; permeabilization and blocking in blocking buffer (0.1% Triton X-100 and 3% FBS in PBS) for 1 h at room temperature; overnight incubation with primary antibodies diluted 1/500 in blocking solution; washing three times in PBS; incubation with Hoechst 33258 (Nacalai Tesque) and secondary antibody diluted 1/500 in blocking solution for 1 h in the dark at room temperature; and washing three times in PBS. Imaging was performed with a Leica AF6000 microscope. The primary antibody mouse anti-CDX2 (MU392A-UC, BioGenex) was used for immunostaining. CF488A donkey anti-mouse IgG (Biotium) secondary antibody was used to visualize signals. For the TUNEL assay, cells were stained with TMR Red using the In Situ Cell Death Detection Kit (Roche) according to the manufacturer's instructions. For the EdU assay, EdU of the Click-iT EdU Imaging Kit (Invitrogen) was added to the ESC culture medium by exchanging half the medium and culturing for 4 h; the cells were then fixed with 4% PFA, permeabilized with 0.1% Triton X-100 in PBS, and stained with 1× Click-iT Reaction Buffer and Hoechst 33258.
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4

Hinokitiol Modulates Cell Signaling Pathways

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Hinokitiol (99%, Figure 1A), dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), collagenase type IV, and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Type I collagen was purchased from Corning (NY, USA). The β‐actin antibody, Dulbecco's modified Eagle's medium (DMEM), L‐glutamine‐penicillin‐streptomycin, fetal bovine serum, Pierce™ 1‐Step Transfer Buffer, SuperScript™ IV First‐Strand Synthesis System Kit, and Fast SYBR™ Green Master Mix were purchased from Thermo Fisher (Waltham, MA, USA). The NucleoSpin® RNA kit was purchased from Macherey‐Nagel (Düren, Germany). Primary antibodies against Bax, Bcl‐2, phospho‐NF‐κB p65 (Ser536), IκBα, cleaved caspase‐3, and PARP were purchased from Cell Signaling (Beverly, MA, USA). The glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody was purchased from GeneTex (Irvine, CA, USA). Hybond‐P polyvinylidene difluoride (PVDF) membrane, the enhanced chemiluminescence (ECL) western blotting detection reagent, horseradish peroxidase (HRP)‐conjugated donkey anti‐rabbit immunoglobulin G (IgG), and the sheep anti‐mouse IgG were purchased from Amersham (Buckinghamshire, UK). CF488A Donkey anti‐mouse IgG and CF594 Donkey anti‐rabbit IgG were purchased from Biotium (Fremont, CA, USA). Hinokitiol was dissolved in 0.1% dimethyl sulfoxide (DMSO) and stored at 4°C.
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5

Embryonic Morphology and Histology Analysis

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Morphological and histological analyses of embryos were collected at various times of gestation. For histological analysis, embryos were rinsed with ice‐cold PBS and fixed with 4% paraformaldehyde at room temperature for 10–30 min, dehydrated through graded alcohols, and embedded in paraffin. Embryos were sectioned at 5 μm thickness and stained with haematoxylin and eosin. Sectioned embryos were photographed using a Nikon E800M inverted microscope. For immunofluorescent analysis, paraffin sections were rehydrated, immunostained with cleaved Caspase‐3 antibody (9664, Cell Signalling Technology), Ki‐67 antibody (12202, Cell Signalling Technology). Antigen retrieval was performed according to the individual antibody instructions. CF488A Donkey anti‐Rabbit IgG (20015, Biotium) and CF488A Donkey anti‐Mouse IgG (20,014, Biotium) were used as the secondary antibody. Nuclei were counterstained with 1 μg/ml Propidium Iodide (P4170, Sigma‐Aldrich), washed and mounted.
Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end‐labeling (TUNEL) assay was performed using MEBSTAIN Apoptosis TUNEL Kit Direct (8445, MBL). Briefly, paraffin‐embedded sections were deparaffinized, DNA nick end‐labelled, and counterstained with 1 μg/ml Propidium Iodide and mounted. The slides were analysed using a TCS SP8 confocal microscope (Leica).
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6

Morphological and Histological Analysis of Embryos

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Morphological and histological analysis of embryos were collected at various times of gestation. For histological analysis, embryos were rinsed with ice-cold PBS and fixed with 4% paraformaldehyde at room temperature for 10-30 min, dehydrated through graded alcohols and embedded in paraffin. Embryos were sectioned at 5 µm thickness and stained with hematoxylin and eosin. Sectioned embryos were photographed using a NIKON microscope. For immunofluorescent analysis, paraffin sections were rehydrated, immunostained with cleaved Caspase-3 antibody (#9664, Cell Signaling), Ki-67 antibody (#12202, Cell Signaling). Antigen retrieval was performed according to the individual antibody instructions. CF488A Donkey anti-Rabbit IgG (#20015, Biotium) and CF488A Donkey anti-Mouse IgG (#20014, Biotium) were used as the secondary antibody. Nuclei were counterstained with 1 g/ml Propidium Iodide (P4170, Sigma-Aldrich), washed and mounted.
Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end labeling (TUNEL) assay was performed according to the manufacturer's instructions (MBL, #8445). Briefly, paraffin-embedded sections were deparaffinized, DNA nick end labelled, and counterstained with 1 g/ml Propidium Iodide and mounted. The slides were analyzed using a TCS SP8 confocal microscope (Leica).
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