PPAR-γ (ab45036), interleukin (IL)-1β (ab9722), tumor necrosis factor-alpha (TNF-α; ab66579), and F4/80 (ab6640) antibodies were obtained from Abcam (Cambridge, UK). CD45 antibody (sc-52491) and rosiglitazone (sc-202795) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). IL-6 (12912S), NF-κB p65 (8242), phospho-NF-κB p65 (3033), stress-activated protein kinase (SAPK)/Jun-amino-terminal kinase (JNK; 9258), phospho-SAPK/JNK (9255S), p38 MAPK (9212L), phospho-p38 MAPK (4631L), p44/42 MAPK (Erk1/2) (4695S), and phospho-p44/42 MAPK (Erk1/2) (9101S) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Invitrogen Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (A-11058); Invitrogen Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (A-11055); and Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody (A-21206) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Horseradish peroxidase (HRP)-conjugated anti-β-actin antibody (a5316) was obtained from Sigma-Aldrich (St. Louis, MO, USA), and 4′,6-diamidino-2-phenylindole (DAPI; H-1200) and mounting medium (H-5000) were obtained from Vector Laboratories (Burlingame, CA, USA).
4 6 diamidino 2 phenylindole dapi h 1200
Manufactured by Vector Laboratories
Sourced in United States
4′,6-diamidino-2-phenylindole (DAPI) (H-1200) is a fluorescent stain that binds to DNA. It is commonly used in various laboratory techniques, such as fluorescence microscopy, to visualize and detect the presence of DNA in cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
F4 80 ab6640, by Abcam (1 mentions)
Phospho p44 42 mapk erk1 2 9101s, by Cell Signaling Technology (1 mentions)
Tnf α ab66579, by Abcam (1 mentions)
Nbp1 31231, by Novus Biologicals (1 mentions)
Ab6564, by Abcam (1 mentions)
Ab14935, by Abcam (1 mentions)
Fluoview fv10i confocal microscope, by Olympus (1 mentions)
Ab150077, by Abcam (1 mentions)
Lsm 510 meta microscope, by Zeiss (1 mentions)
Rabbit polyclonal anti il 1β, by Abcam (1 mentions)
Rabbit anti p62 sqstm1, by Abcam (1 mentions)
Mouse anti 6e10, by BioLegend (1 mentions)
Rabbit gmf polyclonal antibody, by Proteintech (1 mentions)
Mouse gmf monoclonal antibodies, by Proteintech (1 mentions)
Rabbit anti lamp1, by Abcam (1 mentions)
Mouse anti nlrp3, by Adipogen (1 mentions)
Ab34710, by Abcam (1 mentions)
Alexa fluor 594 conjugated donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
16 protocols using 4 6 diamidino 2 phenylindole dapi h 1200
1
Molecular Signaling Pathway Analysis
2
Quantifying Intestinal MUC2 Expression
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Fixed ileum tissues were embedded in paraffin and 5-μm sections were prepared. The sections were incubated overnight in rabbit anti-MUC2 (NBP1-31231, Novus Biologicals, Littleton, CO, USA) at 4 °C. Then, the sections were washed with PBS three times and incubated in fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (bs-0295G, Bioss) for 1 h at room temperature in the dark. For visualization of cell nuclei, 4′,6-diamidino-2-phenylindole (DAPI) (H-1200, Vector Laboratories) was added. Images were captured under a fluorescent microscope (Nikon) at a magnification of 200×. Positively stained cells were counted in all villi for each tissue section.
3
Immunofluorescence Staining of Ly6G and CXCR2
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The sections were deparaffinized with xylene, dehydrated with ethanol, and then heated in 0.01 M citrate buffer (pH 6.0). After blocking with 3% fetal bovine serum (FBS) (in PBS) for 60 min at room temperature, antibodies against Ly6G (1:100, LS-C139872, LSBio, Washington, USA) and CXCR2 (1:100, ab14935, Abcam, Cambridge, UK) were applied overnight at 4 °C. The next day, goat anti-rabbit immunoglobulin G (IgG) H+L (Alexa Fluor® 488) (1:400, ab150077, Abcam, Cambridge, UK) and goat anti-rabbit IgG H+L (Cy5®) (1:400, ab6564, Abcam, Cambridge, UK) secondary antibodies were incubated at 37 °C for 2 h. The slides were mounted using mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (H-1200, Vector Laboratories, Burlingame, CA, USA) to obtain nuclear staining. Images of the cells were taken on a FLUOVIEW confocal microscope (FV10i, Olympus America, Melville, NY, USA).
4
Nrf2 Localization in C6 Cells
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C6 cells (4 × 104) were treated with Tau-Cl (200 μM) for 3 or 9 h or with taurine (200 μM) for 9 h, and then fixed with 4% paraformaldehyde for 20 min. The anti-Nrf2 antibody (16396-1-AP, 1:200; Proteintech) was incubated overnight, followed by incubation with FITC-labeled anti-IgG antibody (AP132F, 1:200; Merck Millipore). Cells were counterstained with 4′,-6-diamidino-2-phenylindole (DAPI; H-1200; Vector Laboratories, Newark, CA, USA) to visualize the nuclei. Images were observed under a Zeiss LSM 510 META microscope (Carl Zeiss Meditec AG, Jena, Germany).
5
Antibody Characterization of Neuroinflammation
6
Immunofluorescence Staining of Decellularized Scaffold
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The decellularized FDM samples were placed on coverslip and fixed by 4% paraformaldehyde for 30 min at room temperature. After being washed with PBS three times, they were blocked using 3% bovine serum albumin (BSA) for 1 h at room temperature. They were incubated with primary antibody solution overnight at 4 °C, then subsequently rinsed with PBS, added with secondary antibody solution, and incubated for 1 h. Lastly, the stained samples were mounted onto microscope glass slides with vectashield® mounting medium containing 4′, 6-diamidino-2-phenylindole (DAPI) (H1200; Vector Lab, Burlingame, CA, USA). Fluorescence images were captured using confocal laser scanning microscope (LSM 700; Carl Zeiss, Oberkochen, Germany). Mouse anti-fibronectin (SC-8422; Santa Cruz Biotechnology, Dallas, TX, USA) and rabbit polyclonal anti-collagen type I (ab34710; Abcam, Waltham, MA, USA) were used as a primary antibody. Secondary antibodies are Alexa Fluor® 488-conjugated goat anti-mouse IgG (A11001; Invitrogen, Waltham, MA, USA) and Alexa Fluor® 594-conjugated donkey anti-rabbit IgG (A21207; Invitrogen).
7
Monocytes Internalize Extracellular Vesicles
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Monocytes (2 × 106/well) were incubated in the presence of PKH67‐labeled EVs ( SW480EV‐S: 6–7 × 109 particles per mL; APEV and RCEV: 5–6 × 1011 particles per mL) for 4 h at 37 °C in a 5% CO2‐humidified environment in 35‐mm glass bottom MatTek dishes (P35G‐1.5‐14‐C; MatTek Corporations, Ashland, MA, USA) coated with Poly‐d ‐Lysine (A‐003‐E; Merck Millipore). The cells were washed with PBS, fixed in 4% paraformaldehyde for 15–20 min at room temperature, and mounted with VECTASHIELD mounting media containing 4',6‐diamidino‐2‐phenylindole (DAPI) (H‐1200; Vector Laboratories, Burlingame, CA, USA). Analysis of EV internalization was performed using a Leica (Leica Microsystem, Wetzlar, Germany) TCS SP8 confocal microscope equipped with a 100 × 1.40 NA oil immersion lens, a UV laser, a continuous wavelength white‐light laser set at 490 nm, and transmitted light. Z‐stack images were acquired for each sample (0.3 µm between z‐planes). Acquired images were further processed using the fiji imagej software (ImageJ, http://imagej.net , W Rasband, National Institute of Health, Bethesda, MD, USA) [19 ]. A PKH67‐PBS sample was included as negative control.
8
Immunofluorescence Staining of KLOTHO in HK-2 Cells
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HK-2 cells were washed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde (Nacalai) and permeabilized with 0.5% Triton X-100 (Nacalai) at room temperature. After blocking with 5% Blocking One Histo (Nacalai) for 10 minutes (min), the cells were incubated with rabbit polyclonal KLOTHO antibody (1:100, PA5-21078, ThermoFisher Scientific) at 37°C for 30 min. After washing with PBS, the cells were incubated with Alexa Flour 488 goat anti-rabbit IgG (1:10,000, Invitrogen) at 37°C for 30 min in the dark. The nucleus was labeled with 4′,6-diamidino-2-phenylindole (DAPI) (H-1200, Vector Laboratories). Images were captured using a Keyence BZ-9000 fluorescence microscope.
9
Immunohistochemical Detection of HIF-1α
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For immunohistochemistry of hypoxia-inducible factor (HIF)-1α, paraffin-embedded sections were deparaffinized in xylene, rehydrated in a graded alcohol series, and immersed in PBS, followed by application of a protein block (Dako) for 10 min. The sections were incubated at 4°C with rabbit polyclonal anti-HIF-1α antibody (1:250; ab2185; Abcam) for 1 h. After extensive washing with PBS, the sections were incubated with Alexa Fluor® 568-conjugated goat anti-rabbit IgG (H+L) secondary antibody (1:500; A-11036; Thermo Fisher Scientific, Inc.) for 30 min at room temperature. After extensive washing with PBS, the sections were counterstained using Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (H-1200; Vector Laboratories).
10
Visualizing Polysialylated Neural Cell Adhesion Molecules in Piglet Hippocampus
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Consecutive coronal sections of 20 μm thickness from the piglet’s hippocampus were prepared using a freezing microtome (LEICA CM 1950, Germany). Brain sections were fixed in ice-cold 4 % paraformaldehyde for 15 min, rinsed in phosphate-buffered saline (PBS), and permeabilized with 0.3 % Triton X-100 for 30 min, followed by blocking with 0.3 % goat serum in PBS (room temperature). The thin sections were incubated overnight (4 °C) with a primary antibody specific for detecting the polySia moiety of polySia-neural cell adhesion molecule (NCAM) (1:200; MAB5324, Millipore, USA). After washing with PBS, sections were incubated with Alexa Fluor 488-conjugated secondary antibody (1:200; 115-545-075, Jackson ImmunoResearch, USA) for 1 h at room temperature. All sections were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) (H-1200, Vector Laboratories, USA). Images were obtained and analyzed using a confocal microscope (FV1000, Olympus, Japan).
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