Total protein was extracted from 6-mm RPE/choroid punches obtained from four donors homozygous for the CFH Y allele, five donors homozygous for the H allele, and five heterozygous donors, and concentrations were determined using the BioRad DC Assay according to the manufacturer’s instructions (BioRad, Hercules, CA, USA). For each sample, 20 μg of protein was diluted in PBS with 2 mm CaCl2 and 140 mm NaCl mixed with Native Sample Buffer (BioRad) and loaded into a 10% Mini-PROTEAN TGX precast gel (BioRad). Semi-native PAGE separation was performed using 0.005% SDS. Proteins were then blotted onto a polyvinylidene difluoride (PVDF) membrane (BioRad). Membranes were treated briefly with methanol followed by blocking with 1% BSA (Research Products International, Mt Prospect, IL, USA) in PBS with 0.1% Tween-20 (PBS-T; Research Products International). To detect both forms of CRP, the membrane was incubated with primary antibodies directed against pCRP (1D6) or mCRP (3H12) for 2.5 h. Following three 5-min washes in PBS-T, membranes were treated with Alexa Fluor® 488-conjugated donkey anti-mouse secondary antibody (A-21202; Thermo Fisher Scientific, Waltham, MA, USA) and washed three times for 5 min each in TBS with 0.1% Tween-20 (TBS-T; BioRad). Finally, bands were detected using a VersaDoc Imager (BioRad).
Alexa fluor 488 conjugated donkey anti mouse secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor 488-conjugated donkey anti-mouse secondary antibody is a fluorescently labeled antibody used for detecting and visualizing mouse primary antibodies in various immunological techniques. It consists of a donkey-derived antibody that binds to mouse immunoglobulins and is conjugated to the Alexa Fluor 488 fluorescent dye, which emits green fluorescence when excited.
Automatically generated - may contain errors
Lab products found in correlation
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Dc assay, by Bio-Rad (1 mentions)
Native sample buffer, by Bio-Rad (1 mentions)
Versadoc imager, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Research Products International (1 mentions)
Mab377, by Merck Group (1 mentions)
Sc 65514, by Santa Cruz Biotechnology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Unc0638, by Selleck Chemicals (1 mentions)
Unc0642, by Selleck Chemicals (1 mentions)
Olaparib, by Selleck Chemicals (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Roti mount fluorcare, by Carl Roth (1 mentions)
Mitotracker red, by Thermo Fisher Scientific (1 mentions)
Laser confocal microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Donkey serum, by Sartorius (1 mentions)
Anti drp1 antibody, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 647 goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 conjugated goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
12 protocols using alexa fluor 488 conjugated donkey anti mouse secondary antibody
1
Quantification of CRP Isoforms in the Retina
2
Quantifying Epigenetic Markers in Prefrontal Cortex
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Immunofluorescence was conducted27 (link) to measure the expression of 5-mC, MeCP2 in neuron cells in the PFC. Primary antibodies raised against NeuN (mouse, 1:500, MAB377; Chemicon International, Inc, Temecula, CA), 5-mC (rabbit, 1:500, #28692; Cell Signaling Technology, Beverly, MA), MeCP2 (rabbit, 1:1,000, #3456; Cell Signaling Technology), and brain-derived neurotrophic factor (mouse, 1:10, sc-65514; Santa Cruz Biotechnology, Santa Cruz, CA) were diluted in 1× PBST(phosphate buffered saline (PBS) + 0.3% Triton X-100) supplemented with 0.3% bovine serum albumin. The plates were wrapped in aluminum foil to block light and then stored at 4°C for 72 hours. The tissue sections were sequentially incubated for 1 hour with a mixture of Alexa Fluor 488-conjugated donkey anti-mouse secondary antibody (1:1000; Thermo Fisher Scientific, Fremont, CA) and Alexa Fluor 594-conjugated donkey anti-goat secondary antibody (1:2000; Thermo Fisher Scientific). Cell counting (NeuN/5-mC and/MeCP2) was performed using a square grid (300 × 300 μm) in the PFC.
3
Immunofluorescence Staining of Fixed Cells
4
Investigating DNA Damage Response
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Olaparib (#S1060), UNC0638 (#S8071), and UNC0642 (#S7230) were obtained from SelleckChem. Full details of antibodies and usage for immunoblotting and immunofluorescence are given in Additional file 4 : Table S3. Alexa Fluor 488-conjugated donkey anti-mouse secondary antibody (Invitrogen #A21202, 1:1000) was used for immunofluorescence detection of γH2AX and H3K9me2.
5
Immunofluorescent Localization of Antioxidant Enzymes
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BRL cells were treated and incubated with H2O2 as described in Section 2.6 . Then, the cells were fixed with 4% (m/v) paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% bovine serum albumin. Thereafter, the cells were stained with the primary antibody (anti-Superoxide Dismutase (SOD) rabbit polyclonal antibody and anti-COX IV mouse polyclonal antibody (Proteintech, USA)) and subsequently with Alexa Fluor 488-conjugated donkey anti-mouse secondary antibody or Alexa Fluor 568-conjugated donkey anti-rabbit secondary antibody (Invitrogen, USA). After cell staining, images were acquired with a confocal laser scanning microscope (Carl Zeis AG, Germany).
6
Quantification of Theileria annulata Schizonts
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To count the number of schizont nuclei in each infected cell, cells were grown on round cover slips (10 mm diameter, Car Roth) in 24-well plates to sub-confluency in absence or presence of BPQ. Cells were then fixed by 4% paraformaldehyde (PFA) (Sigma) in PBS for 10–15 min. The cover slips were then fixed on microscope slide with a drop of mounting medium containing DAPI (ROTI®Mount FluorCare, Carl Roth, Germany). Parasite nuclei were quantified manually under a Leica DMi8 wide field microscope. To evaluate BPQ effect on T. annulata schizonts, BPQ treated or control TaC12 cells were first fixed with 4% paraformaldehyde (PFA) (Sigma-Aldrich) for 10 min and stained with mouse polyclonal anti-Theileria p104 antiserum (Gift of Brian Shiels, University of Glasgow, 1:1000 dilution in 0.4% Triton 100, 1% BSA in PBS) for 1 h. Following three PBS washes, Alexa Fluor 488 conjugated donkey anti-mouse secondary antibody (Invitrogen) (1:500 dilution in 0,1% Triton 100 in PBS for 1 h) was used to visualize p104 signal under a wide field fluorescent microscope (Leica DMi8). Presence of any residual p104 signal was considered as the cell being p104 positive. All staining steps were performed at room temperature. When necessary, images were taken by LASX PC software and finally processed in Fiji Image J.
7
Mitochondrial Morphology Analysis in PC12 Cells
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PC12 cells were seeded onto coverslips and incubated for 24 h. After treatment, the cells were incubated with 100 nmol/L MitoTracker Red (ThermoFisher Scientific, Waltham, MA, USA) at 37°C for 30 min. Next, the cells were fixed in 4% PFA for 30 min and permeabilized with 0.3% Triton X-100 in PBS for 10 min at room temperature. The cells were blocked with 10% donkey serum (Biological Industries, Cromwell, CT, USA) in PBS for 30 min at 37°C and then incubated with anti-Drp1 antibody (1:50, Santa Cruz Biotechnology) overnight at 4°C. Subsequently, the cells were incubated with Alexa Fluor 488-conjugated donkey anti-mouse secondary antibody (1:200, Invitrogen, Carlsbad, CA, USA) for 1 h at 37°C, and then counterstained with DAPI; (Beyotime) and visualized under a laser confocal microscope (Leica, Wetzlar, Germany). Mitochondrial morpholo2gical characteristics, including form factor (FF, perimeter2/[4π×area]) and aspect ratio (AR, major axis/minor axis), were quantified using ImageJ software (NIH, Bethesda, MD, USA) as described before.[29 (link)]
8
Immunofluorescence Visualization of Cellular Proteins
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The transfected HeLa or HEK293T cells were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100/PBS for 10 min, and then blocked with 5% FBS/PBS for 1 h. MxB-FLAG was detected by incubation with mouse anti-FLAG antibody (Sigma, catalog F1804-200UG, 1:1000) for 2 h, followed by incubation with Alexa Fluor 488 conjugated donkey anti-mouse secondary antibody (Invitrogen, catalog A21202, 1:500) for 1 h. TNPO1 was detected using mouse anti-TNPO1 antibody (Abcam, catalog ab10303, 1:1000) for 2 h, then with Alexa Fluor 647-goat anti-mouse secondary antibody (Invitrogen, catalog A21237, 1:500) for 1 h. KPNB1 was detected with mouse anti-KPNB1 antibody (Invitrogen, catalog MA3-070, 1:500) for 2 h and Alexa Fluor 647 conjugated goat anti-mouse secondary antibody (Invitrogen, catalog A21237,1:500) for 1 h. DNA was stained with DAPI (4=,6-diamidino-2-phenylindole). Images were recorded with a Quorum WaveFX Spinning Disk Confocal using a 63x oil objective.
9
Integrin α5β1 Localization in Cell Lines
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C666-1, HONE-1, and HK-1 cells were initially grown on cover slips. The coverslips were then transferred to 35 mm culture dishes and treated with ICG-001 for 7 days. Then, the medium was discarded and PBS was used to rinse the cells for three times. Cells were fixed with 4% paraformaldehyde for 10 min. The cell-containing 35 mm dishes were then placed on an ice bath and incubated with primary and secondary antibodies. Afterwards, the cells were fixed with a DAPI-containing Mountant (ProLong Gold Antifade Mountant, Thermofisher, #P36930). The confocal images of cells stained with DAPI, integrin α5β1 (antibody from Millipore, Clone JBS5, #MAB1969) with AlexaFluor-488 conjugated donkey anti-mouse secondary antibody (Invitrogen, #A21202), and Phalloidin (antibody from ThermoFisher, #A12381) were captured under confocal microscopy at an excitation wavelength of 405, 495, and 595 nm, respectively.
10
Immunofluorescence Staining of PANC-1 Cells
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PANC-1 cells were fixed with 4% paraformaldehyde at 4 °C overnight and then washed with PBS in 5 min for three times. Fixed cells were blocked in antibody dilution buffer (PBS containing 0.25% Triton X-100 and 1% bovine serum albumin), and all subsequent staining was performed in the same buffer. The cells were labeled with primary antibody for 2 h and subsequently with Alexa Fluor 488-conjugated donkey anti-mouse secondary antibody (Invitrogen Life Technologies) for 1.5 h. The nucleus was stained by incubation with DAPI for 15 min at room temperature. Cells were scanned using a ZEISS LSM 800 confocal microscope (Zeiss, Jena, TH, GER) equipped with a 63 × objective.
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