The protein samples from HEK293T cells transfected with the expression vector WT1 wild type or mutant were diluted with Laemmli sample buffer (Bio-Rad Laboratories, Inc., Hercules, CA) and boiled for 10 minutes. Samples were loaded into 4% to 20% Mini-PROTEAN TGX Precast Protein gels (Bio-Rad Laboratories, Inc.) and electrophoresed in Tris-glycine-SDS running buffer. Proteins were transferred to Immun-Blot PVDF Membrane (Bio-Rad Laboratories, Inc.), in accordance with standard protocols, after which membranes were blocked with Block Ace (KAC Co., Ltd., Kyoto, Japan). Membranes were incubated overnight at 4°C with 10× Block Ace and rabbit monoclonal antibodies against WT1 ab89901 (Abcam, Cambridge, UK) or β-actin (Cat. No. 4967; Cell Signaling Technology, Danvers, MA). Subsequently, blots were washed and incubated for 1 hour at room temperature with goat anti-rabbit IgG antibody and HRP-conjugated secondary antibody. Immunoreactive proteins were visualized with Immobilon Western Chemiluminescent HRP Substrate (Millipore) followed by luminescence detection with ChemiDoc MP (Bio-Rad).
Block ace
Manufactured by Abcam
Sourced in United Kingdom
Block Ace is a laboratory product designed to prevent non-specific binding in immunoassays. It is a protein-based blocking buffer that can be used to minimize background signals and improve the specificity of antibody-based detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
Immun blot pvdf membrane, by Bio-Rad (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Chemidoc mp, by Bio-Rad (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Block ace, by Sumitomo Pharma (1 mentions)
Cy3 conjugated secondary antibody, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Fluoview fv1000 microscope, by Olympus (1 mentions)
Fuluorshield with dapi, by Vector Laboratories (1 mentions)
Prl monoclonal antibody, by Abcam (1 mentions)
3 protocols using block ace
1
Western Blot Analysis of WT1 Protein
2
Immunofluorescence Staining of Brain Sections
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The brain was perfused with cold saline and fixed with phosphate buffer (pH 7.4) containing 4% formaldehyde. Coronal brain sections (8 μm thick) were incubated with blocking buffer (4% w/v, Block Ace; Dainippon Sumitomo Pharma Co. Ltd., Osaka, Japan) for 2 h. The sections were incubated with polyclonal rabbit anti-cleaved caspase-3 or RIP3 antibody (1 : 100, Abcam, Cambridge, UK) in 1% w/v Block Ace overnight at 4°C. After washing with PBS, these were correspondingly incubated with Cy3-conjugated secondary antibody (1 : 100, Chemicon, Temecula, CA, USA) for 2 h at room temperature. Finally, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA) for 15 min at room temperature, washed, and mounted using a mounting medium with 80% glycerol. Immunofluorescence was visualized using the fluorescence microscopy as described above. The percentage of cleaved caspase-3-positive cells was calculated as the number of the positive-staining nuclei to the total nuclei [33 (link), 40 (link)]. Regarding RIP3, because a lower level of expression of this protein was observed in every cell in the hippocampus, the cells having an enhanced expression of RIP3 under a laser beam at constant intensity were determined as RIP3-positive. All histopathological scoring and evaluation was performed by an investigator without knowledge of the treatment.
3
Immunofluorescent Detection of PRL and PAK2
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Prior to immunohistochemistry, the tissue sections were deparaffinized in xylene and ethanol. Sections were treated with Block Ace for 1 h at room temperature
and incubated with PRL monoclonal antibody (Abcam, Cambridge, UK) at a 1:5000 dilution over night at 4ºC. Then, the sections were incubated with a cy3
conjugated-anti rabbit secondary antibody at 1:500 dilution for 1 h and treated with 10% rabbit serum for 1 h. After blocking, the sections were incubated with
an alexafluor 647 conjugated-PAK2 antibody at 1:250 dilution for 1 h at room temperature. Coverslips were mounted in FULUORSHIELD with DAPI (Vector
laboratories, Burlingame, CA, USA) and images were acquired on a FLUOVIEW FV-1000 microscope (Olympus, Tokyo, Japan). Ten fields were selected randomly from
each section under a 200 × field, and the PRL, PAK2 and DAPI positive cells in the field were quantified using ImageJ software (NIH, Bethesda, MD, USA).
and incubated with PRL monoclonal antibody (Abcam, Cambridge, UK) at a 1:5000 dilution over night at 4ºC. Then, the sections were incubated with a cy3
conjugated-anti rabbit secondary antibody at 1:500 dilution for 1 h and treated with 10% rabbit serum for 1 h. After blocking, the sections were incubated with
an alexafluor 647 conjugated-PAK2 antibody at 1:250 dilution for 1 h at room temperature. Coverslips were mounted in FULUORSHIELD with DAPI (Vector
laboratories, Burlingame, CA, USA) and images were acquired on a FLUOVIEW FV-1000 microscope (Olympus, Tokyo, Japan). Ten fields were selected randomly from
each section under a 200 × field, and the PRL, PAK2 and DAPI positive cells in the field were quantified using ImageJ software (NIH, Bethesda, MD, USA).
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