Twenty-micrometer serial section of mouse half-brain sample were collected with cryotome at -20°C. Slices were incubated in citrate buffer (sodium citrate at 0.1 M and 10% ethanol, pH = 8.5) during 40 min at 70°C before 30 min at room temperature (RT). Then, slices were incubated in hydroxyperoxide during 30 min and sodium borohydrure 0.1% during 30 min. Slices were put in blocking solution (9% NBS, 1% BSA, and 0.5% Triton and PBS) during 1 h at RT. Finally, slices stayed over-night at 4°C in blocking solution with primary antibody: Iba1 (1:500, #019-19741, Wako), NeuN (1:500, MAB377, Millipore), MAP2 (1:500, AB5622, Millipore), or GFAP (1:500, clone SMI22, #835301, BioLegend). On the second day, the slices stayed 2 h at RT before an incubation of 1 h 30 min at RT in the secondary antibody solution (Alexa Fluor 568 anti-mouse #1736975, anti-rabbit #1832035, Alexa Fluor 488 anti-rabbit #A11034, anti-mouse #A11029) followed by 7 min in DAPI (D3571, ThermoFisher). Slices were then mounted on lamella and cover-slipped with Fluoromount mounting media (Invitrogen, Thermo Fisher Scientific; #00-4958-02). Slices were observed using a Zeiss AxioImager M2 microscope, zoom 10X/20X and images were processed with a computerized image analysis system (ZEN 2012 SP2 Software, Zeiss).
D3571
Manufactured by Thermo Fisher Scientific
Sourced in United States
The D3571 is a laboratory instrument designed for high-throughput sample preparation. It features an automated liquid handling system capable of precise and accurate pipetting of a wide range of sample volumes. The core function of the D3571 is to automate repetitive liquid handling tasks, improving efficiency and reducing the risk of human error in the laboratory workflow.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (17 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (4 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (4 mentions)
Lsm 710, by Zeiss (4 mentions)
Ab15580, by Abcam (3 mentions)
Fv1000, by Olympus (3 mentions)
Ab13970, by Abcam (3 mentions)
P6148, by Merck Group (3 mentions)
T8787, by Merck Group (3 mentions)
Phalloidin 488, by Thermo Fisher Scientific (2 mentions)
Em grade formaldehyde, by Polysciences (2 mentions)
Schneider s media, by Genesee Scientific (2 mentions)
Slowfade antifade, by Thermo Fisher Scientific (2 mentions)
Nanog rcab002p, by ReproCELL (2 mentions)
Ab192256, by Abcam (2 mentions)
Ab9661, by Cell Signaling Technology (2 mentions)
Alexa 647, by Thermo Fisher Scientific (2 mentions)
H 3300, by Vector Laboratories (2 mentions)
α actin fitc acta2, by Merck Group (2 mentions)
Irak phosphor t209, by Abcam (2 mentions)
103 protocols using d3571
1
Immunohistochemical Staining of Mouse Brain
2
Immunohistochemistry of FFPE Tissue Sections
3
Single-Cell Isolation and Clonal Expansion
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Seventy-two hours after transfection, cells were detached by trypsin (25200056; Life Technologies) and counted with a countess automated cell counter (Invitrogen). Ten thousand cells were resuspended in PBS (1X; pH 7.4; 10010-023; Thermo Fisher Scientific, Inc) containing 20% fetal bovine serum (Atlanta Biologicals) and treated with DNA-staining DAPI (D3571; Thermo Fisher Scientific, Inc) prior to sorting by FACS with a BD FACSymphony S6 Cell Sorter (BD Sciences). Forward scatter (FSC)-area and side scatter (SSC)-area settings were used to gate for intact cells and exclude debris. For singlet isolation, the samples were gated by SSC-height and SSC-width. To further discriminate against doublet events, the cells were subsequently gated using FSC-height and FSC-width parameters. Following debris and doublet exclusion, live singlets were gated using 4′,6-diamidino-2-phenylindole in the BV421-A channel and FSC-area parameters were used to exclude dead cells. The identified live singlet population was then analyzed and sorted for double positive eGFP- and mScarlet-expressing cells. Single cells were sorted into 96-well plates (3596; Costar) prefilled with 100 μl of media using FITC-A and PE-Texas Red-A settings. The selected cells were then incubated at 37 °C in a humidified environment of 5% CO2 for a minimal of 6 weeks, changing media every 2 weeks.
4
Isolation and Staining of Mammoth Muscle Nuclei
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Buffer A was prepared by mixing 98 μl of the nuclear isolation media (731086; Beckman Coulter) and 2 μl of 0.05% trypsin-EDTA solution (25300-054; Thermo Fisher Scientific). Mammoth muscle tissue (approximately 30 mg) was minced with forceps and scissors in a culture dish. Next, the minced muscle tissue was transferred into a BioMasher column, followed by the addition of 100 μl Buffer A and homogenisation using the BioMasher III (320302; Nippi). After centrifugation at 900 g for 4 min, the supernatant was transferred into a new 0.2 ml tube, the same volume of TE buffer (pH 8.0) was added and mixed well, and the mixed solution was centrifuged at 400 g for 4 min. The supernatant was removed and the pellet was suspended into 30 μl of CZB medium. DAPI (D3571; Thermo Fisher Scientific) was added to the nuclei suspension at a final concentration of 0.1 µg/ml. The suspension was transferred into a new culture dish and somatic cell nuclei with intrinsic fluorescence were picked up under a fluorescent microscope for nuclear transfer.
5
Biolistic Transformation of Onion Epidermis
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DNA coating and bombardment of onion epidermis were performed by the Biolistic® PDS-1000/He Particle Delivery System (Bio-Rad) according to the manufacturer’s protocol. A total of 0.75 mg of 10 µm gold particles (Bio-Rad) and 1.25 µg of plasmid DNA were used for each bombardment event. Onion epidermal cells were treated with DAPI (D3571, ThermoFisher Scientific) to stain the nuclei and observed using a confocal microscope, Olympus FV1000 [excitation 488 nm, emission 510 nm for green fluorescent protein (GFP); and excitation 405 nm, emission 461 nm for DAPI]. The images were processed by FV10-ASW 4.2 Viewer.
6
Fluorescent Cell Cytoskeleton Labeling
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Phalloidin 488 (1:500; Thermo Fisher Scientific, A12379). Nuclear staining: minimum 30 minutes incubation in DAPI (Thermo Fisher Scientific, D3571) + PBS (5mg/ml).
7
Immunolabeling of Frozen Skin Biopsies
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Fresh frozen skin biopsy tissues were cryo-sectioned into 8 µm slices and then fixed and permeabilized in acetone for 20 min at -20°C. After air dry and PBS wash, the slides were incubated with primary antibodies overnight at 4°C and followed by PBS wash, 1-hour incubation with fluorescence-labeled secondary antibodies at room temperature, PBS wash, counterstained with DAPI (Thermo Fisher Scientific Cat# D3571, RRID: AB_2307445) and mounted in Prolong Gold Antifade Mountant (Thermo Fisher Scientific, Cat# P36930). The primary antibodies used in this study were specific for human NCAM (BioLegend Cat# 304602, RRID: AB_314444), CD8 (BD Biosciences Cat# 557708, RRID: AB_314070), CD4 (BioLegend Cat# 300502, RRID: AB_314070), IFI16 (Abcam Cat# ab55328, RRID: AB_2121692), HSV2 (Agilent Cat# B0116, RRID: AB_2335703).
8
Immunostaining of Drosophila Ovaries
9
Quantitative Immunofluorescence Analysis of LDLR
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Cells were grown in 24-well-plates to reach confluence after 48 h. Following a washing step with PBS, cells were heat-fixed at 80°C for 4 h. For staining of LDLR, cells were incubated with either the monoclonal LDLR antibody (ab52818, Abcam; diluted 1:500 in PBS + 0.05% Tween) or the polyclonal LDLR antibody (10785-1-AP, Proteintech; diluted 1:500) at 37°C for 1 h, followed by three washing steps and incubation with secondary antibody goat anti-rabbit IgG-Cy3 (111-165-003, dianova; diluted 1:1,000) at 37°C for 1 h. After another three washes, cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; D3571, Thermo Fisher Scientific; diluted 1:500).
For immunofluorescence (IF) analysis, a Leica DMI3000 B microscope with a Leica DFC3000 G camera (Leica Microsystems) and the Leica Application Suite software (version 4.13.0) were employed. For quantitative comparison of the LDLR expression of different cell lines, the experiment was repeated three times, and each time five pictures per well were taken. Using the ImageJ software (version 1.51q), images were converted to HSB stacks and the total brightness was measured. The average brightness per cell was calculated using the DAPI staining as reference.
For immunofluorescence (IF) analysis, a Leica DMI3000 B microscope with a Leica DFC3000 G camera (Leica Microsystems) and the Leica Application Suite software (version 4.13.0) were employed. For quantitative comparison of the LDLR expression of different cell lines, the experiment was repeated three times, and each time five pictures per well were taken. Using the ImageJ software (version 1.51q), images were converted to HSB stacks and the total brightness was measured. The average brightness per cell was calculated using the DAPI staining as reference.
10
Immunofluorescence Analysis of Epithelial Markers
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MEECs were grown to an appropriate density and fixed in 4% (w/v) paraformaldehyde for 15 min, permeabilized with 0.5% Triton-X-100 for 15 min, and blocked with 5% bovine serum albumin for one hour at room temperature. After blocking, cells were labeled with the primary antibodies (1:100, rabbit anti-EpCAM, Proteintech, 21050-1-AP; 1: 100, rabbit anti-Mucin1, Abcam, ab109185; 1:100, mouse anti-P63, Abcam, ab735; 1:100, rabbit anti-CD44, Proteintech, 15675-1-AP; 1:100, mouse anti-ERα, Santa Cruz Biotechnologies, sc-71064; 1:100, mouse anti-PR, Santa Cruz Biotechnologies, sc-398898; 1:100, rabbit anti-Vimentin, Abcam, ab137321), and the secondary antibodies (1:100, fluorescently labeled goat anti-mouse lgG-cy3, BA1031, Boster company, Wuhan, China) according to the manufacture’s protocol. DAPI (0.5 mg/ml, D3571, Thermo Fisher) was used to stain the nucleus. Then, the fluorescence was detected by Leica DM4000B fluorescence microscope.
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