Immunohistochemistry and immunofluorescent staining were performed according to the standard procedures (28 (link)). Paraffin-embedded colon tumor tissues sections were prepared and epitope retrieval was performed for further analysis. The paraffin sections were treated with hydrogen peroxide (3%) for 10–15 min, which subsequently blocked by a regular blocking solution for 10–15 min 37°C for immunohistochemistry. Colon tumor cells were cultured and stained with microtubule associated protein 1 light chain 3 α (MAP1LC3A), translocase of outer mitochondrial membrane 20 (TOMM20) for observation of microtubules and/or microvessels, Neo (Nase), and calreticulin (Invitrogen; Thermo Fisher Scientific, Inc.), NRP-2 (ab129050; Abcam), Apaf-1 (ab2001; Abcam), Bad (ab32445; Abcam) for immunofluorescent staining. All antibodies were used at a dilution of 1:1,000. Cells were then incubated with goat anti-rabbit IgG H&L (HRP) (1:2,000; ab205718; Abcam) for 1 h at 37°C. All fluorescent samples were visualized with a confocal fluorescence microscope (Leica TCS SP8; Leica Corporation, Wetzlar, Germany).
Ab2001
Manufactured by Abcam
Sourced in United States
Ab2001 is a laboratory equipment product. It serves a core function within the lab setting.
Automatically generated - may contain errors
Lab products found in correlation
Ab32124, by Abcam (3 mentions)
Ab32445, by Abcam (2 mentions)
Ab205718, by Abcam (2 mentions)
Ab9485, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab152126, by Abcam (1 mentions)
Ab32522, by Abcam (1 mentions)
Ab69643, by Abcam (1 mentions)
Ab4051, by Abcam (1 mentions)
Ab2014, by Abcam (1 mentions)
Enhanced chemiluminescence reagent, by Bio-Rad (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Ab92742, by Abcam (1 mentions)
Ab32042, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab32351, by Abcam (1 mentions)
Ab133504, by Abcam (1 mentions)
Ab32371, by Abcam (1 mentions)
Ab92552, by Abcam (1 mentions)
5 protocols using ab2001
1
Immunostaining of Colon Tumor Tissues
2
Apoptosis Signaling Proteins in Podocytes
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Protein expression levels of E2F3, Bcl-2 related X protein (Bax), B-cell
lymphoma-2 (Bcl-2), Bad, apoptotic peptidase activating factor 1 (APAF1),
C-caspase3, C-caspase7, and C-caspase9 were measured using western blot assay in
podocytes under different treatments. Transfected podocytes (2×106cells/well) were lysed using pre-cold RIPA buffer (Beyotime Institute of
Biotechnology) according to the manufacturer instructions. Podocyte lysates were
separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), and then isolated proteins were transferred onto a polyvinylidene
fluoride (PVDF) membrane (Millipore, USA). After blocking the proteins with 5%
non-fat milk in the membranes for 1 h at room temperature, membranes were
incubated with primary antibodies against E2F3 (ab152126, 1:2000 dilution), Bad
(ab32445, 1:1000 dilution), Bcl-2 (ab32124, 1:1000 dilution), Bax (ab69643,
1:1000 dilution), APAF1 (ab2001, 1:5000 dilution), C-caspase3 (ab4051, 1:1000
dilution), C-caspase7 (ab32522, 1:1000 dilution), C-caspase9 (ab2014, 1:1000
dilution), and GAPDH (1:1000, ab9485) (all from Abcam, UK) overnight at 4°C. The
horseradish peroxidase (HRP)-linked secondary antibody (ab205718, 1:10,000
dilution, Abcam) was then incubated with the membranes at room temperature for 1
h. Finally, an ECL detection kit (Pierce Biotechnolgy, USA) was used to detect
the protein bands.
lymphoma-2 (Bcl-2), Bad, apoptotic peptidase activating factor 1 (APAF1),
C-caspase3, C-caspase7, and C-caspase9 were measured using western blot assay in
podocytes under different treatments. Transfected podocytes (2×106cells/well) were lysed using pre-cold RIPA buffer (Beyotime Institute of
Biotechnology) according to the manufacturer instructions. Podocyte lysates were
separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), and then isolated proteins were transferred onto a polyvinylidene
fluoride (PVDF) membrane (Millipore, USA). After blocking the proteins with 5%
non-fat milk in the membranes for 1 h at room temperature, membranes were
incubated with primary antibodies against E2F3 (ab152126, 1:2000 dilution), Bad
(ab32445, 1:1000 dilution), Bcl-2 (ab32124, 1:1000 dilution), Bax (ab69643,
1:1000 dilution), APAF1 (ab2001, 1:5000 dilution), C-caspase3 (ab4051, 1:1000
dilution), C-caspase7 (ab32522, 1:1000 dilution), C-caspase9 (ab2014, 1:1000
dilution), and GAPDH (1:1000, ab9485) (all from Abcam, UK) overnight at 4°C. The
horseradish peroxidase (HRP)-linked secondary antibody (ab205718, 1:10,000
dilution, Abcam) was then incubated with the membranes at room temperature for 1
h. Finally, an ECL detection kit (Pierce Biotechnolgy, USA) was used to detect
the protein bands.
3
Western Blot Analysis of Apoptotic and Proliferative Markers in MDA-MB-468 Cells
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The treated MDA-MB-468 cells were lysed with the cell lysis buffer. Protein concentration was measure by the BCA Protein Assay kit (Beyotime Biotechnolgy, Beijing). Lysates were separated in 10% SDS-PAGE and then transferred onto the nitrocellulose membrane (Millopore, USA). The membrane was blocked with 5% skimmed milk in TBST buffer at room temperature for 1 h. After that, the membranes were incubated with anti-Caspase 3 (1:5000, ab32351, Abcam, Cambridge, MA, USA), anti-Cleaved Caspase 3 (1:500, ab32042, Abcam), anti-Ki67 (1:5000, ab92742, Abcam), anti-PCNA (1:2000, ab92552, Abcam), anti-Bak (1:10000, ab32371, Abcam), anti-Bax (1:1000; ab32503, Abcam), anti-Bcl-2 (1:1000, ab32124, Abcam), anti-apaf-1 (1:1000, ab2001, Abcam), anti-Cytochrome c (1:5000; ab133504, Abcam), anti-Akt (1:1000; #4685, Cell Signaling Technology, Danvers, MA), anti-phosphorylated-Akt (1:1000, #4060 CST), anti-p38 (1:1000, #8690, CST), anti-phosphorylated p38 (1:1000, #4511, CST), anti-NF-κB (1:1000, #8242, CST), anti-p21 (1:1000; ab109520, Abcam), anti-p27 (1:5000; ab32034, Abcam) and GAPDH (1:2500, ab9485, Abcam) at 4°C overnight and then secondary antibodies (1:5000) were incubated for 1 h at room temperature. The proteins were visualized by using the enhanced chemiluminescence reagent (Bio-Rad, USA).
4
Immunohistochemical Analysis of Apoptosis Markers
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From tumors of each treatment group, 4-μm-thin tissue sections were cut using a microtome and transferred to gelatin-coated slides [36 (link)]. Tissue sections were incubated with primary antibodies anti-FADD (ab24533), anti-apoptotic protease activating factor 1 (APAF-1) (ab2001), and BCL-2 (ab32124; Abcam, Burlingame, CA, USA) at 4 °C overnight. Slices were washed with phosphate-buffered saline (PBS) and incubated with a streptavidin/Haptoglobin Related Protein (HRP)-conjugated secondary antibody (Biocare Medical, Concord, CA, USA). Immunoreactivity to the various proteins was visualized with a colorimetric-based detection kit following the protocol provided by the manufacturer (TrekAvidin-HRP Label + Kit from Biocare Medical, Pacheco, USA). Light microscopy (Nikon Eclipse 2000 equipped with Nikon DS-Fi2; Nikon Corporation, Tokyo, Japan) with a high-power objective (40×) was used to acquire digital images. The intensity of cell immunostaining was scored as follows: 1 = absence of positive cells, 2 = small number of positive cells or isolated cells, 3 = moderate number of positive cells, and 4 = large number of positive cells. Labelling intensity was evaluated by two previously trained examiners in a double-blind fashion.
5
Immunofluorescence Analysis of APAF1 in miR-186 Modulated A-431 Cells
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miR-NC-, miR-186 mimic- or miR-186 inhibitor-transfected A-431 cells were seeded at a density of 1×105 cells/ml on a coverslip pre-coated with poly-L-lysine. They were subsequently fixed with cold 4% formaldehyde at 4°C overnight. After washing three times with PBS containing 0.1% Triton X-100, cells were blocked with 10% bovine serum albumin (cat. no. FA016-50G; Amresco, LLC, Solon, OH, USA) for 2 h at RT followed by incubation with primary antibodies against APAF1 (1:1,000; cat. no. ab2001; Abcam) and DAPI (1:2,000; cat. no. ab104139; Abcam) at 4°C overnight. Cells were then incubated with Alexa Fluor 488 donkey anti-mouse immunoglobulin G (1:200; ab150105; Abcam) secondary antibodies for 1 h at RT and visualized using a confocal laser-scanning microscope. Magnification at ×200.
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