Immunocytochemistry protocol was based on a previously published report (16 ). In brief, cultured mouse and human DRG neurons were fixed with 10% formalin (Thermo-Fisher, 23–245684) at room temperature for 10 minutes followed by 3 washes of 1X PBS. Cells were subsequently blocked for 1 hour at room temperature with 10% normal goat serum (NGS, R&D systems S13150H) dissolved in 1X PBS-Tx (0.02% Triton X-100 in 1X PBS, Sigma-Aldrich X100–5ML). Cells were incubated overnight at 4°C in primary antibody dissolved in antibody solution (2% NGS and 2% bovine serum albumin (BSA) dissolved in PBS-Tx). Cells were washed three times with 1X PBS. Secondary antibodies were also dissolved in antibody solution. Cells were incubated in secondary antibodies for 1 hour at room temperature followed by three 1X PBS washes. DAPI (Cayman Chemicals, 14285) was dissolved in 1X PBS and washed twice with 1X PBS. Human DRGs were incubated with TrueBlack (Biotium 23007) for 30 seconds to quench lipofuscin signal. Coverslips were mounted onto slides before being imaged. Confocal images were obtained on Olympus FV-1200 microscope at 10X magnification. Antibodies used in this study were: p-eIF2α (1:500, Cell Signaling #3398), β3 tubulin (1:1000, Sigma #T8578), DAPI (1:10,000, Cayman Chemicals #14285), Alexa Fluor 488 (1:500, Life Technologies, A11034), and Alexa Fluor 555 (1:500, Life Technologies, A21428).
4 6 diamidino 2 phenylindole (dapi)
Manufactured by Cayman Chemical
Sourced in United States
DAPI (4',6-diamidino-2-phenylindole) is a fluorescent dye used for staining and visualizing nucleic acids, particularly DNA, in biological samples. It binds to the minor groove of DNA, emitting a blue fluorescence when excited by ultraviolet (UV) light. DAPI is commonly used in various applications, such as fluorescence microscopy, flow cytometry, and nucleic acid quantification.
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Lab products found in correlation
Lsm 710, by Zeiss (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Evos 2 fl, by Thermo Fisher Scientific (2 mentions)
Hcs studio cell analysis software, by Thermo Fisher Scientific (2 mentions)
Poly d lysine coated glass bottom dishes, by MatTek (2 mentions)
Fluorescence microscopy, by Zeiss (2 mentions)
Anti mouse igg h l alexa fluor 594 secondary antibody, by Abcam (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Facscelesta flow cytometer, by BD (2 mentions)
F4 80, by BioLegend (2 mentions)
Donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
α sma, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Dp80 microscope, by Olympus (1 mentions)
Cellsens 1, by Olympus (1 mentions)
Glass chamber slides, by BD (1 mentions)
Mowiol, by Merck Group (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
22 protocols using 4 6 diamidino 2 phenylindole (dapi)
1
Immunocytochemistry of DRG Neurons
2
Co-Staining of Lung Tissue Fibroblasts
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Co-staining was performed on lung tissue sections and fibroblasts. Samples were incubated in 10% donkey serum (92590, Temecula, CA) for 30 min at 37 °C. After co-incubation overnight at 4 °C with Gαi3 (sc-365422, Santa Cruz Biotechnology, Dallas, TX)/α-SMA, PKA(ADI-KAS-PK017, Enzo, Farmingdale, NY)/α-SMA, p-CREB/α-SMA and p-Smad2/3 (ART1568, Antibody Revolution, San Diego, CA)/α-SMA, sections were incubated with products from Novex (Life Technologies, Frederick, MD): donkey anti-rabbit IgG (H + L) FITC (A16024), donkey anti-mouse IgG (H + L) TRITC (A16016), Alexa Fluor 647 donkey anti-goat IgG (H + L) (A21447) or donkey anti-rabbit IgG (H + L) TRITC (A16028) and donkey anti-mouse IgG (H + L) FITC (A16018) for 60 min each at 37 °C in blocking buffer. Nuclei were stained with DAPI (14285, Cayman, Ann Arbor, MI) for 5 min. Cells or tissues were visualized under an Olympus DP80 microscope and were analyzed with image software (Cell Sens 1.8, Olympus Corporation, Germany).
3
Immunofluorescence Assay for CRTC1
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HEK293T cells were plated in glass chamber slides (BD) and forward transfected with Lipofectamine® 2000 (Invitrogen) using mCRTC1 constructs in the pUbC-3xHA backbone. Roughly 24 h post-transfection cells were treated with either DMSO or 10 μM Forskolin for 30 min. Slides were fixed with 4% paraformaldehyde and incubated with primary antibodies (HA.11, CRTC1). Samples were incubated with secondary antibodies conjugated with Alexa Fluor® -488 (donkey anti-mouse) and -568 (donkey anti-rabbit) (Life Technologies) and subsequently counterstained with DAPI (Cayman Chemical Company) before images were acquired with a LSM 780 (Carl Zeiss).
4
Staphylococcus aureus Phagocytosis Assay
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Macrophages were stimulated with vehicle DMSO (Sigma Aldrich, D2650) or WY14643 for 60 minutes, then biological triplicates were infected at an MOI of 10, 20, or 50 with heat-killed Staphylococcus aureus Newman-CFP for 30, 60, 90, or 120 minutes. 24 well plates were immediately centrifuged at 200 x g for 5 minutes. Macrophages were fixed at end of experimental trial and stained with DAPI (Cayman Chemical, 14285, 300nM), then imaged using the EVOS 2 FL (Invitrogen) and analyzed with HCS Cell Studio Analysis Software (ThermoFisher) to detect fluorescent bacteria within stained macrophages.
5
Immunofluorescence Staining of Melanocytes
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NB1RGB cells or B16 melanoma cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The epidermal sheets and skin sections described above were fixed in ice-cold 95% ethanol for 30 min. Each sample was immersed in a blocking solution containing 10% newborn calf serum in PBS for 60 min at room temperature and incubated with HA-M-INK lysates (or control COS-7 cell lysates) in the blocking solution at 4 °C overnight. After washing three times with PBS, they were incubated with anti-HA tag antibody (1:400 dilution), anti-Pmel antibody (HMB45; 1:400 dilution), anti-c-Kit antibody (1:100 dilution), or anti-desmoplakin 1 (DP-1) antibody (1:400 dilution) at room temperature for 60 min. The samples were then washed three times with PBS, incubated with DAPI (Cayman Chemical, Ann Arbor, MI, USA) and anti-rat/mouse/rabbit/guinea pig Alexa Fluor 488/568/647 IgG at room temperature for 60 min, and mounted in Mowiol (Merck Millipore).
6
Immunohistochemical Tissue Preparation Protocol
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Slides were removed from the cryostat and immediately transferred to cold 10% formalin (4°C; pH 7.4) for 15 minutes. The tissues were then dehydrated in 50% ethanol (5 min), 70% ethanol (5 min), 100% ethanol (5 min), 100% ethanol (5 min) at room temperature. The slides were air dried briefly and then boundaries were drawn around each section using a hydrophobic pen (ImmEdge PAP pen, Vector Labs). When hydrophobic boundaries had dried, the slides were submerged in blocking buffer (10% Normal Goat Serum, 0.3% Triton-X 100 in 0.1M Phosphate Buffer (PB) for 1 hour at room temperature. Slides were then rinsed in 0.1M PB, placed in a light-protected humidity-controlled tray and incubated in primary antibodies diluted in blocking buffer overnight at 4°C. The next day, slides were washed in 0.1M PB and then incubated in their respective secondary antibody diluted at 1:2000 with DAPI (1:5000; Cayman Chemical; Cat 14285) in blocking buffer for 1 hour at room temperature. The antibodies used are provided in Table 2 . The sections were washed in 0.1M PB and then covered with True Black (20% diluted in 70% Ethanol), a blocker of lipofuscin, for 1 minute. Sections were then washed in water, air dried and coverslipped with Prolong Gold Antifade reagent (Cat P36930, Fisher Scientific).
7
Fluorescence imaging of SIK-CRTC signaling
8
Immunocytochemical Analysis of HaCaT Cells
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The HaCaT cells were seeded in six-well confocal plates at a density of 1 × 106/well. After 24 h of incubation, the cells were washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 5 min at 37 °C. Then, the cells were incubated with 5% bovine serum albumin dissolved in PBS for 45 min at 37 °C. The HaCaT cells were incubated overnight with one of the following primary antibodies for immunocytochemistry: OR2AT4 (PA5-39811, 1:200; Thermo Fisher Scientific), sodium/potassium-ATPase α (Na+/K+-ATPase-α) (SC-48345, 1:50; Santa Cruz Biotechnology), and Ki-67 (NB500-170, 1:200; Novus). Then, the cells were washed three times with PBS. The HaCaT cells were incubated with anti-mouse IgG (Alexa Fluor 488; A11001, 1:500; Thermo Fisher Scientific) or anti-rabbit IgG (Alexa Fluor 532; A11009, 1:500; Thermo Fisher Scientific) secondary antibody for 1 h at room temperature. Next, the cells were incubated with 300 nM DAPI (Cayman Chemical, Ann Arbor, MI, USA) for 5 min in the dark. The cells were washed twice with PBS. Images were obtained using the LSM510 META confocal microscope and analyzed using LSM700 software (version 3.2; Carl Zeiss, Jena, Germany).
9
Epigenetic Modulation of Cellular Processes
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Antibodies include: Abcam: anti-H3K27me3 (ab192985); Cell Signaling: anti-H3K9me3 (13,969), anti-H4K20me3, anti-γH2AX (9718); Roth: DAPI (6843.1) Compounds include: Cayman; Valproic Acid (13,033), CHIR99021 (13,122), Repsox (14,794), Forskolin (11,018), Doxorubicin (15,007); Acros Organics: TCP (130472500); APExBIO: DZNep (A8182); Seleckchem: TTNPB (S4627); Roth: X-beta-Gal (2315.3), Ascorbic Acid (Sigma).
10
Lysosomal Integrity and Cell Viability Assay
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Lysosomal integrity and the viability of cells was assessed using LysoTracker Red DND (Invitrogen, USA) and DAPI (Cayman Chemical, Ann Arbor, MI, USA), respectively. The CLL cells (1 × 106 cells/mL) were treated with indicated compounds for 24 h, then the culture medium was removed (centrifugation, 200× g, 5 min), and the cells were resuspended in 500 µL 100 nM LysoTracker Red DND-99. The cells were loaded with the probe for 1 h at 37 °C, then 3 µM DAPI was added for 10 min. The cells were then pelleted, resuspended in 20 µL culture medium, and analyzed using an imaging flow cytometer (Amnis® ImageStream®X Mk II; Luminex Corporation, Austin, TX, USA). A minimum of 10,000 events in focus was collected per sample. Data are presented as means ± SEM of 3 independent experiments.
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