The antibodies used were as follows: anti-Flag M2 (F3165, Sigma-Aldrich at 1:500 for immunofluorescence (IF) and 1:1,000 for western blotting (WB), anti-Emerin (5430S, Cell Signalling Technology at 1:500 for IF), anti-Actin Ab-5 (612,656, Bioscience International at 1:3,000 for WB). For IF, secondary antibodies; Alexa Fluor 488 (A32766, Molecular Probes, 1:300) and 594 (A32754, Molecular Probes, 1:300). Secondary antibodies used for WB were Donkey anti-Mouse 800 nm (IRDye 800CW 926-32212, LiCor, 1:5,000), Donkey anti-Rabbit (IRDye 680LT 926-28023, LiCor, 1:5,000).
A32754
Manufactured by Thermo Fisher Scientific
Sourced in United States
The A32754 is a laboratory equipment designed for measuring and analyzing samples. It is a precision instrument used for various applications in scientific research and testing environments.
Automatically generated - may contain errors
Lab products found in correlation
A32723, by Thermo Fisher Scientific (4 mentions)
A32766, by Thermo Fisher Scientific (3 mentions)
A32814, by Thermo Fisher Scientific (3 mentions)
Ab61682, by Abcam (2 mentions)
Widefield microscope celldiscoverer7 cd7, by Zeiss (2 mentions)
D9564, by Merck Group (2 mentions)
Sc 15408, by Santa Cruz Biotechnology (2 mentions)
A21208, by Thermo Fisher Scientific (2 mentions)
A32744, by Thermo Fisher Scientific (2 mentions)
Ab104224, by Abcam (2 mentions)
Ab109223, by Abcam (2 mentions)
Ab25877, by Abcam (2 mentions)
H3570, by Thermo Fisher Scientific (2 mentions)
Tcs sp8, by Leica (2 mentions)
Irdye 800cw 926 32212, by LI COR (1 mentions)
Anti flag m2 f3165, by Merck Group (1 mentions)
Gtx128145, by GeneTex (1 mentions)
Ab10983, by Abcam (1 mentions)
Mca497r, by Bio-Rad (1 mentions)
Lsm 980, by Zeiss (1 mentions)
18 protocols using a32754
1
Immunofluorescence and Western Blotting Protocols
2
Antibody Characterization for DNA Damage Response
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The antibodies against ATR (2790), Chk1 (2345), Ser345 phospho-Chk1 (2341), RPA32 (52448), XPC for immuno-precipitation assay (14768) and Histone H3 (9715) were purchased from Cell Signaling. IntS3 (A302-051A) and Ser33 phospho-RPA32 (A300-246A) antibodies were purchased from Bethyl Laboratories. Antibodies against XPC for immuno-fluorescence assay (GTX70308) and Ser1989 phospho-ATR (GTX128145) were purchased from Genetex. CPD (T1192) and gamma-tubulin (T6557) antibodies were purchased from Sigma. Antibodies against histone H2B (ab1790) and hSSB2 (16419-1-AP) were purchased respectively from Abcam and ProteinTech. The hSSB1 antibody was purified from sheep anti-serum as described previously31 (link). Fluorescent secondary antibodies used were: Donkey anti-Mouse 800 nm (LI-COR; IRDye 800CW 926–32,212, 1:5000 for WB), Donkey anti-Rabbit (LI-COR; IRDye 680LT 926–28,023, 1:5000 for WB) and Alexa Fluor 488 (A32766, Molecular Probes, 1:200 for IF) and 594 (A32754, Molecular Probes, 1:200 for IF).
3
Comprehensive Histology and Staining Protocol
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Tissues were fixed in 10% formalin, subjected to paraffin-embedded sectioning and stained with hematoxylin or immunostained with anti-UCP1 (Abcam, ab10983, 1:100) and anti-perilipin A (Abcam, ab61682. 1:100) antibodies followed by incubation with the secondary antibody conjugated with Alexa 488 (Invitrogen, A32814, 1:500) and 594 (Invitrogen, A32754, 1:500). TUNEL (terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling) (Takara, cat. no. MK500), cleaved caspase-3 (Cell signaling, #9661, 1:100), Iba-1 (Wako, 019-19741, 1:200), and F4/80 (Serotec, MCA497R, 1:200) immunostaining were performed per manufacturer’s instructions. To assess hepatic steatosis, multiple slides of frozen liver sections fixed with 10% buffered formalin for 30 min at room temperature were stained for 7 min with a filtered solution of 0.7% Oil Red O in propylene glycol, and counterstained with hematoxylin for 1 min, washed 3 times with distilled water, and then visualized. Liver sections were also stained with azan to identify fibrosis. To estimate adipocyte size, tissues (iWAT and eWAT) were fixed overnight with neutral-buffered 10% formalin at 4 °C, paraffin embedded, sectioned, and stained with hematoxylin and eosin. Adipocyte area was determined by quantifying the adipocyte area of a total of 20000~ cells from 7–9 animals per study group using Adiposoft image analysis software.
4
Immunofluorescence Staining of ATRX in 3D Cell Cultures
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800 C3H/10T1/2 cells were seeded per well in a 96 well plate (glass bottom culture plates, MatTek, PBK96G-1.5-5-F) and kept at 37-degree Celcius for 24 h. Cells were fixed with 4% formaldehyde in PBS for 10 min at room temperature. Cells were permeabilized with 0.2% digitonin (EMD Millipore, 300410) in PBS (Corning, 21-040-CV) for 10 min at room temperature. Next, the cells were incubated in 3% BSA (in PBS, Sigma, #9418) for blocking for 1 h at room temperature followed by incubation in primary antibody (ATRX, Santa Cruz, sc-15408, 1:200) at 4°C overnight, followed by secondary antibody (1:600, Invitrogen, A32754) incubation at room temperature for 1 h. Cells were washed three times with PBS for 5 min followed by DAPI staining (2 μg/ml in PBS, Sigma, D9564) for 5 min at room temperature. Mounting media (Vectashield, H-1000) was added immediately after DAPI staining. Cells were imaged using Widefield Microscope CellDiscoverer7 (CD7) automated widefield high-throughput system (Zeiss). Images were processed with ImageJ software (http://rsb.info.nih.gov/ij/ ). For the ATRX antibody, the ImageJ Brightness/Contrast was set as 30/112. For DAPI, the Brightness/Contrast was set as 37/123. All images were processed with the same parameter settings for each antibody.
5
Immunostaining of Vascular Markers
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Mice hearts and cornea were embedded in OCT (SAKURA, Torrance, CA, USA). Immunostainings were performed on 7 μm-thick cryosections or on HUVECs on coverslips. 2% PFA was used to fix tissue sections or cells. Primary antibodies and dilutions: CD31 (1:100, ab281583, Abcam, Cambridge, UK), VEGF165 (1:250, ab52917, Abcam, Cambridge, UK) and VEGFR2 (1:500, 9698, CST, Danvers, MA, USA). Fluorescently labeled Alex 594 secondary antibody (1:200, A32754, A48278, Invitrogen, Waltham, MA, USA) were used. DAPI visualized cell nucleus. Fluorescent signals were detected with a confocal laser scanning microscope (LSM980, Zeiss, Oberkochen, Germany).
6
Neuroinflammation and Alzheimer's Pathology
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Mice were euthanized and perfused with 4% paraformaldehyde (PFA), and whole brains were carefully removed and fixed by immersion in PFA, followed by dehydration with 30% sucrose. The brains were OCT-embedded to obtain 8-μm-thick frozen sections. For the staining procedure, brain slices were incubated in a blocking solution (10% donkey serum, 0.3% Triton 100 in PBS solution) at room temperature for 1 h. After PBS washes, the sections were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: Iba-1 (ab283346; Abcam) and BACE1 (ab183612; Abcam). After PBS washes, sections were incubated with fluorescent secondary antibody antibodies: donkey anti-rat Alexa Fluor 488 (A48269TR; Invitrogen) and donkey anti-rabbit Alexa Fluor 594 (A32754; Invitrogen). Finally, the brain slices were washed 3 times with PBS and then incubated with DAPI Fluoroshield mounting medium in the dark, followed by photography using confocal microscopy. All the images were taken at the peri-hematoma area.
7
Immunostaining of Embryonic Gonad Sections
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Embryonic gonads were fixed in 4% PFA for 30 min at 4°C. Gonads were next submerged in 10 and 20% sucrose in PBS for 1 h each at 4°C, and in 30% sucrose in PBS overnight at 4°C. The gonads were then embedded in Tissue-Tek O.C.T. compound (Sakura Finetek) and frozen in liquid nitrogen. Six-micrometer-thick sections of each gonad were applied to glass slides and autoclaved in TRS (Dako). After pre-incubation with 3% skim milk in PBS-T (PBS with 0.1% Tween20 (Sigma-Aldrich)) at RT for 30 min, the sections were reacted with primary antibodies overnight at 4°C at the following dilutions: 1:200 for mouse anti-phospho-histone H3 (ser10) (1:200, Sigma-Aldrich, 06-570), goat anti-CDH1 (1μg/ml, R&D, AF748), rabbit anti-DNMT3L (1:500, provided by Dr. Yamanaka), rabbit anti-STRA8 (1:200, abcam #ab49602), rabbit anti-Ki67 (1:200, Invitrogen #MA5-14520), rat anti-Ki67 (1:100, Invitrogen AB_10854564) and anti-pS6 (1:200, CST #2211). Secondary antibodies labeled with Alexa 488, 594 or 647 (1:1,000, Invitrogen A21207, A21208, A21209, A32766, A32814, A32754, A32744 and A32795) were used. DNA was counter-stained with DAPI (100 ng/ml). Fluorescence microscopy was performed using Olympus FV1200 and images were processed with ImageJ/Fiji (Schindelin et al., 2012 (link)).
8
Immunofluorescence Imaging of Brain Tissue
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Brain tissue was postfixed in 4% paraformaldehyde, dehydrated with sucrose solution and sliced to 10 μm. Cultured neurons were fixed with 4% paraformaldehyde. Then they were permeabilized with 0.3% Triton X-100, and blocked with immunostaining blocking solution (Epizyme, Shanghai, China). After incubation with diluted primary antibody overnight at 4 ℃, the membranes were washed 3 times for 10 min with PBS and Tween 20. The antibodies were as follows: anti-PDK4 (1:200, 89295, Abcam), anti-NeuN (1:200, 26975-1-AP, Proteintech, Rosemont, IL, USA), anti-Iba1 (1:200, 5076, Abcam), anti-GFAP (1:200, 4648, Abcam). The next day, they were incubated with corresponding secondary antibodies: anti-rabbit Alex Fluor 488-conjugated secondary antibody (1:200, A11008, Invitrogen), anti-rabbit Alexa Fluor 594-conjugated secondary antibody (1:200, A32754, Invitrogen), anti-mice Alexa Fluor 594-conjugated secondary antibody (1:200, A32740, Invitrogen), anti-mice Alexa Fluor 488-conjugated secondary antibody (1:200, A32723, Invitrogen). Immunofluorescence images were captured by using the microscope (ZEISS, HB050, Berlin, Germany) and analyzed with ImageJ.
9
Immunohistochemical Analysis of Neuronal Markers
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Mice were anesthetized with sevoflurane, and brains were cut on a sliding freezing stage microtome in 30 μm thick sections. Hippocampal sections were washed 3 times in PBS. Following 15 min permeabilization with 0.3% Triton X-100 in 0.01 M PBS, sections were blocked with 10% donkey serum for 2 h. Next, sections were incubated overnight at 4°C with the following primary antibodies: DHX9 (1:300, 17721-1-AP, Proteintech), NeuN (1:200, ab104224, Abcam), Iba1 (1:200, ab5076, Abcam), GFAP (1:200, ab3554, Abcam), MAP2 (1:200, ab11267, Abcam), FMRP (1:100, sc-101048, Santa Cruz), STAU1 (1:100, sc-390820, Santa Cruz), or c-Fos (1:200, ab208942, Abcam). The next day, slides were washed 3 times for 7 min in PBS. Then, sections were incubated with the corresponding fluorescent-conjugated secondary antibody (A32766, A32744, A32790, A32754, Invitrogen) for 2 h at room temperature in the dark. DAPI Fluoromount-G aqueous mounting medium with anti-fade properties (SouthernBiotech) was used to identify cell nuclei. Slides were stored in the dark at 4°C until needed for analysis. Fluorescence images were captured with an Olympus FV1000 laser confocal microscope. For c-Fos positive neuron analysis, ImageJ was used to analysis the signals of hippocampal neurons from 3 animals per condition.
10
Quantifying ATRX Protein Expression
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800 C3H/10T1/2 cells were seeded in a 96 well plate (glass bottom culture plates, MatTek, PBK96G-1.5-5-F) and keep cells in 37-degree for 24 h. Cells were fixed with 4% formaldehyde in PBS for 10 minutes at room temperature. Cells were permeabilized with 0.2% digitonin (EMD Millipore, 300410) in PBS (Corning, 21-040-CV) for 10 minutes at room temperature. Next, the cells were incubated in 3% BSA (in PBS, Sigma, #9418) for blocking for 1 h at room temperature followed by incubation in primary antibody (ATRX, Santa Cruz, sc-15408, 1:200) at 4-degree overnight, followed by secondary antibody (1:600, Invitrogen, A32754) incubation at room temperature for 1 h. Cells were washed three times with PBS for 5 minutes followed by DAPI staining (2 μg/ml in PBS, Sigma, D9564) for 5 minutes at room temperature. Mounting media (Vectashield, H-1000) was added immediately after DAPI staining. Cells were imaged using Widefield Microscope CellDiscoverer7 (CD7) automated widefield high-throughput system (Zeiss). Images were processed with ImageJ software (http://rsb.info.nih.gov/ij/ ). For the ATRX antibody, the ImageJ Brightness/Contrast was set as 30/112. For DAPI, the Brightness/Contrast was set as 37/123. All images were processed with the same parameter settings for each antibody.
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