We used a freezing microtome (Thermo Scientific, Waltham, MA, USA) to obtain coronal sections (15 μm thickness) from the brain tissue with ischemic lesions. The slices were then permeated with 0.5% Triton X-100 for 20 min, blocked with 10% donkey serum at 37 °C for 1 h, and incubated overnight with primary antibodies at 4 °C. The primary antibodies were as follows: rabbit anti-NLRP3 (1:200, Bioworld Technology, Nanjing, China); rabbit anti-Caspase-1 (1:100, Affinity biosciences, Changzhou, China); and rabbit anti-IL-1β (1:100, Abcam, Waltham, MA, USA). The following day, brain slices were washed three times with phosphate-buffered saline (PBS) and incubated with secondary antibodies for 1 h at 37 °C. After three additional washes with PBS, they were treated with Fluoromount-G containing 4′,6-diamidino-2-phenylindole (DAPI; Southern Biotech, Birmingham, AL, USA) to stain the nuclei. Immunofluorescence was observed using a laser scanning confocal microscope (Zeiss LSM880, Oberkochen, Germany). ImageJ software was used to measure the number of positive cells in five different areas around the cerebral infarction [25 (link)].
Fluoromount g containing 4 6 diamidino 2 phenylindole dapi
Manufactured by Southern Biotech
Sourced in United States
Fluoromount-G is an aqueous, permanent mounting medium that contains 4′,6-diamidino-2-phenylindole (DAPI), a fluorescent stain that binds to DNA. It is used for mounting and preserving fluorescently labeled samples for microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880, by Zeiss (1 mentions)
Freezing microtome, by Thermo Fisher Scientific (1 mentions)
Rabbit anti caspase 1, by Affinity Biosciences (1 mentions)
Rabbit anti il 1β, by Abcam (1 mentions)
Alexa 647, by Thermo Fisher Scientific (1 mentions)
Pipes, by Merck Group (1 mentions)
Ix70 microscope, by Olympus (1 mentions)
Cellsens software, by Olympus (1 mentions)
Dp80 camera, by Olympus (1 mentions)
Evos cell imaging system, by Thermo Fisher Scientific (1 mentions)
Dp71 camera, by Olympus (1 mentions)
Desmin, by Abcam (1 mentions)
Ix50 microscope, by Olympus (1 mentions)
α sma, by Agilent Technologies (1 mentions)
Itga5, by Abcam (1 mentions)
Itgb1, by Abcam (1 mentions)
5 protocols using fluoromount g containing 4 6 diamidino 2 phenylindole dapi
1
Immunofluorescence Analysis of NLRP3, Caspase-1, and IL-1β in Cerebral Infarction
2
Localization of snRNAs and P-bodies
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Cy3-labeled DNA probes were used against U1, U2 and U4 snRNAs as previously described (18 (link)):
Forty-eight to seventy-two hours after siRNA transfection, cells were fixed in 4% paraformaldehyde/PIPES (Sigma) for 15 min, permeabilized with 0.5% Triton X-100 for 5 min and incubated with anti-DDX6 antibodies as a marker of P-bodies followed by incubation with secondary antibody conjugated with Alexa-647 (Life Technologies). Cells were again fixed in 4% paraformaldehyde/PIPES for 5 min, quenched for 5 min in 0.1 M glycine/0.2 M Tris, pH 7.4, and incubated with Cy3-labeled probes in 2× SSC/50% formamide/10% dextran sulfate/1% bovine serum albumin for 60 min at 37°C. After washing in 2× SSC/50% formamide, 2× SSC and 1× SSC, coverslips were mounted to microscope slides using Fluoromount G containing 4′,6-diamidino-2-phenylindole (DAPI) (Southern Biotech) for DNA staining. Images were collected using a DeltaVision microscope system as described earlier.
Forty-eight to seventy-two hours after siRNA transfection, cells were fixed in 4% paraformaldehyde/PIPES (Sigma) for 15 min, permeabilized with 0.5% Triton X-100 for 5 min and incubated with anti-DDX6 antibodies as a marker of P-bodies followed by incubation with secondary antibody conjugated with Alexa-647 (Life Technologies). Cells were again fixed in 4% paraformaldehyde/PIPES for 5 min, quenched for 5 min in 0.1 M glycine/0.2 M Tris, pH 7.4, and incubated with Cy3-labeled probes in 2× SSC/50% formamide/10% dextran sulfate/1% bovine serum albumin for 60 min at 37°C. After washing in 2× SSC/50% formamide, 2× SSC and 1× SSC, coverslips were mounted to microscope slides using Fluoromount G containing 4′,6-diamidino-2-phenylindole (DAPI) (Southern Biotech) for DNA staining. Images were collected using a DeltaVision microscope system as described earlier.
3
Quantification of QUAD 3.0-WP936 Conjugate Binding
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First, 1 × 104 U-251 GBM cells were plated in each well of an 8-well chamber slide and allowed to adhere overnight. QUAD 3.0-WP936 conjugate was added in various amounts and incubated at 37 °C. Wells treated with PBS served as a control. After fixation with 10% buffered formalin, cells were permeabilized with PBS/1% BSA/0.1% Triton-X 100 for 10 min at 37 °C. QUAD 3.0 protein was detected by staining with Alexa-488-conjugated anti-human IgG (ThermoFisher Scientific, Waltham, MA) for 1 h at room temperature. Slides were washed with PBS and mounted with Fluoromount-G containing 4’,6-diamidino-2-phenylindole DAPI (Southern Biotech, Birmingham, AL, UK). WP936 fluoresces in the TRITC channel (excitation: 544 nm, emission: 570 nm). Images were captured on an Olympus IX70 microscope with a DP80 camera and compiled in CellSens software (Olympus, Waltham, MA, USA).
4
Cardiac Fibroblast Wound Healing Assay
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Cardiac fibroblasts were seeded at a density of 12,000 cells/well in a 96-well plate, maintained at 37°C and 5% CO2 overnight and starved the next day with 1% EV-depleted FBS medium for 24 h. The monolayer was scratched with a 200 µl pipette tip as described previously [25 ,26 ]. Cells were washed with PBS and incubated with 5 × 108 EVs/ml in 1% EV-depleted FBS medium for 24 h. PBS was used as negative control. For evaluation of the scratch, cells were fixed with 4% (v/v) formaldehyde, washed with PBS containing 0.05% Tween 20, permeabilized with 0.1% (v/v) Triton X-100 and stained with Fluoromount-G containing 4’,6-diamidino-2-phenylindole (DAPI, SouthernBiotech Birmingham, Al, USA). Images of stained cells were taken with the EVOS cell imaging system (Thermo Fisher Scientific, Waltham, MA, USA). Scratches were quantified by using ImageJ and the MRI_Wound_Healing_Tool (NIH, Bethesda, MD, USA).
5
Immunofluorescence Imaging of Rat Liver Samples
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Cryosections of young, old, regenerating (PHx), and perfused rat livers as well as isolated HSC under static and shear stress conditions were fixed in ice‐cold methanol or 4% formalin (n = 3). The following primary antibodies were used: COL1 (C2456; Sigma‐Aldrich), COL4 (ab6586; Abcam), LAMA5 (NBP2‐42391; Novus Biologicals), FN (610078; Becton Dickinson), ITGA5 (ab150361; Abcam), ITGB1 (ab52971; Abcam), HGF (80429‐R052; Sino Biological), GFAP (MAB3402; Merck/Millipore), desmin (ab8592; Abcam), and α‐SMA (M0851; Dako/Agilent). The samples were then incubated with anti‐mouse or anti‐rabbit secondary antibodies labeled with Cy3 or FITC (Millipore). The tissue sections were mounted with Fluoromount G containing 4′,6‐diamidino‐2‐phenylindole (DAPI; Southern Biotech) and a glass coverslip. Images were taken using an Olympus IX50 microscope equipped with a DP71 camera (Olympus). The same exposure time was used for each antibody to enable comparison of liver sections from different age groups.
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