The expression of VEGF and Claudin 5 in bEnd.3 cells was confirmed by immunocytochemistry. Cells in all experimental groups were washed three times with PBS, fixed with 4% paraformaldehyde for 3 h, and then washed with PBS. bEnd.3 cells were permeabilized with 0.025% Triton X-100 and blocked for 1 h at RT with dilution buffer (Invitrogen, CA, USA). Primary anti-rabbit VEGF (1 : 500, Millipore, MA, USA) and anti-rabbit Claudin 5 (1 : 500, Santa Cruz, CA, USA) antibodies were prepared in dilution buffer, added to samples, and incubated for 3 h at RT. Primary antibody was then removed and cells were washed three times for 3 min each with PBS. Later, samples were incubated with FITC-conjugated goat, anti-rabbit (1 : 200, Jackson Immunoresearch, PA, USA) or Rhodamine-conjugated donkey, or anti-rabbit secondary antibodies (1 : 500, Millipore, MA, USA) for 2 h at RT. Cells were washed again three times for 3 min each with PBS and stained with 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) (1 : 100, Invitrogen, CA, USA) for 10 min at RT. Fixed samples were imaged using a Zeiss LSM 700 confocal microscope (Carl Zeiss, NY, USA).
Dilution buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
Dilution buffer is a laboratory reagent used to dilute samples or other materials in various analytical and experimental procedures. It serves to maintain the appropriate pH, ionic strength, and other properties of the diluted sample, ensuring accurate and consistent measurements or reactions. The core function of dilution buffer is to facilitate the controlled dilution of samples without affecting their essential characteristics.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700 confocal microscope, by Zeiss (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Fitc conjugated goat anti rabbit, by Jackson ImmunoResearch (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Biotin elution buffer, by Thermo Fisher Scientific (1 mentions)
Pierce nucleic acid compatible streptavidin magnetic beads, by Thermo Fisher Scientific (1 mentions)
Dnarelease additive, by Thermo Fisher Scientific (1 mentions)
6 protocols using dilution buffer
1
Immunocytochemical Analysis of VEGF and Claudin 5
2
Immunocytochemistry of N2A Cell Markers
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The expression of BDNF, cleaved caspase-3, Bcl2, and PSD-95 in N2A cells was confirmed by immunocytochemistry. Cells in all experimental groups were washed three times with PBS, fixed with 4% paraformaldehyde for 3 h, and then washed with PBS. N2A cells were permeabilized with 0.025% Triton X-100 and blocked for 1 h with dilution buffer (Invitrogen, Carlsbad, CA, USA). The following primary antibodies: anti-rabbit BDNF (1 : 500, Abcam, Cambridge, MA, USA), anti-rabbit cleaved caspase-3 (1 : 500, Santa Cruz, Santa Cruz, CA, USA), anti-rabbit PSD-95 (1 : 500, Millipore, Massachusetts, MA, USA), anti-mouse Bcl2 (1 : 500, Millipore, Massachusetts, MA, USA) were prepared in dilution buffer, added to samples, and incubated for 3 h. Primary antibody was then removed and cells were washed three times for 3 min each with PBS. Later, samples were incubated with FITC-conjugated goat, anti-rabbit (1 : 200, Jackson Immunoresearch, PA, USA), or rhodamine-conjugated donkey, anti-mouse secondary antibodies (1 : 500, Millipore, Massachusetts, MA, USA) for 2 h. Cells were washed again three times for 3 min each with PBS and stained with 1 μg/mL DAPI (1 : 100, Sigma-Aldrich, St. Louis, MO, USA) for 10 min at room temperature. Fixed samples were imaged using a Zeiss LSM 700 confocal microscope (Carl Zeiss, Thornwood, NY, USA).
3
Immunocytochemical Analysis of 8-OHdG and Claudin 5
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The expression of 8-Oxo-2'-deoxyguanosine (8-OHdG) and Claudin 5 in bEND.3 cells was confirmed by immunocytochemistry. All the experimental groups was washed 3 times with PBS, fixed with 4% paraformaldehyde for 3 hours, and then washed with PBS. bEND.3 cells were permeabilized with 0.025% Triton X-100 and were blocked for 1 hour at room temperature with dilution buffer (Invitrogen, CA, USA). Primary antibody anti-rabbit 8-OHdG (1:500, Santa Cruz, CA, USA), anti-rabbit Claudin 5 (1:500, Millipore, MA, USA) prepared in the dilution buffer was added to the samples and incubated for 3 hours at room temperature. Primary antibody was removed and cells were washed 3 times for 3 min each with PBS. Later samples were incubated with FITC-conjugated goat anti rabbit second antibodies (1:200, Jackson Immunoresearch, PA, USA) for 2 hours at room temperature. Cells were washed again 3 times for 3 min each with PBS and stained with 1 µg/ml 4',6-diamidino-2-phenylindole (DAPI) (1:100, Invitrogen, CA, USA) for 10 minutes at room temperature. The fixed samples were imaged using Zeiss LSM 700 confocal microscope (Carl Zeiss, NY, USA).
4
Beclin1 and ATG3 Expression in BV2 Cells
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The expression of Beclin1 and ATG3 in BV2 cells was examined by immunocytochemistry. Cells in all experimental groups were washed three times with PBS, fixed with 4% paraformaldehyde for 3 h, and then washed with PBS. BV2 cells were permeabilized with 0.025% Triton X-100 and blocked for 1 h at room temperature with dilution buffer (Invitrogen, Carlsbad, CA, USA). Primary antibodies, anti-rabbit-Beclin1 (1 : 500, Santa Cruz, CA, USA), and anti-rabbit-ATG3 (1 : 500, Santa Cruz, CA, USA) were prepared in dilution buffer, added to samples, and incubated for 3 h at room temperature. Primary antibody was then removed, and cells were washed 3 times for 3 min each with PBS. Next, samples were incubated with Rhodamine-conjugated goat anti-rabbit (1 : 200, Jackson Immunoresearch) for 1 h 30 min at room temperature. Cells were washed again three times for 3 min each with PBS and stained with 1 µg/mL 4',6-diamidino-2-phenylindole (DAPI) (1 : 100, Invitrogen, Carlsbad, CA, USA) for 15 min at room temperature. Fixed samples were imaged using a Zeiss LSM 700 confocal microscope (Carl Zeiss, Thornwood, NY, USA) [49 (link)].
5
Biotin-RNA Pull-Down Proteomics
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For RNA pull-down experiment, 1×107 cells were collected and resuspended in ice-cold PBS and washed. A total of 400 µL Dilution buffer (Thermo Scientific) was added for full lysis, together with a cocktail of protease inhibitors, phosphatase inhibitors and RNase inhibitors (Invitrogen). Then, 150 µL of Pierce Nucleic-Acid Compatible Streptavidin Magnetic Beads (Thermo Scientific) were taken, the beads were washed twice with wash buffer and the stock solution was removed with RNA capture buffer. Beads were incubated for 15–30 minutes with labeled circRBM33 biotin-RNA. The beads were washed five times with wash buffer and the proteins were eluted using 50 µL Biotin Elution buffer (Thermo Scientific). Finally, the retrieved proteins were used for mass spectrometry or western blot analysis.
6
Quantifying CRISPR Activity in HEK293T Cells
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For measuring activity in mammalian cells, HEK293T cells are seeded at a density of 15,000 cells per well (96-well plate) 1 day before transfection. Cells are transfected with 140 ng effector plasmid and 60 ng guide RNA plasmid per well. After 72 h, cells are trypsinized using TrypLE, and 100 μL phosphate buffered saline (PBS) is added to each well. Ten to 20 μL are transferred to a new plate for crude DNA extraction (using Dilution Buffer and DNA Release Additive from Thermo Fisher). One microliter of crude DNA is used in a barcoded Amplicon PCR for NGS preparation using the 300 bp single end kit, target depth of 20,000 reads. Reads are analyzed for InDel formation using CRISPResso.38 (link) For ribonucleoprotein (RNP) testing, a similar procedure was performed, nucleofecting 104 pmol of protein and 120 pmol sgRNA per well.
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