U87 cells were washed three times with PBS and fixed with 4% formaldehyde in PBS for 15 min. After washing with PBS, cells were permeabilized with Triton X-100 (0.5%) at room temperature for 5 min., which was followed by two washing steps with 1% BSA in PBS. Then, cells were incubated with an anti-γ-H2AX primary antibody (mouse anti-γ-H2AX (ser139), Stressgen, bioreagents Corp., Canada) at 2 μg/mL for 1 h. After being washed twice with 1% BSA in PBS, cells were incubated with a FITC-conjugated anti-mouse secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1 mg/mL for 1 h, followed by three washing steps with PBS. After incubation with Hoechst (Sigma-Aldrich, St. Louis, MO, USA) at 1 μg/mL for 5 min, cells were mounted in anti-fade mounting media (Vectashield H-100, Vector Laboratories, Burlingame, CA, USA).
Vectashield h 100
Manufactured by Vector Laboratories
Sourced in United States
Vectashield (H-100) is a mounting medium manufactured by Vector Laboratories. It is designed to be used for the preservation and visualization of fluorescent signals in microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Bx60 epifluorescence microscope, by Olympus (2 mentions)
Metamorph software, by Molecular Devices (2 mentions)
Cytospin 4, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Hoechst, by Merck Group (1 mentions)
Fitc conjugated anti mouse secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Confocal fluorescence microscope, by Leica (1 mentions)
Eclipse ti e fluorescence microscope, by Nikon (1 mentions)
Pco edge camera, by Nikon (1 mentions)
R960 25, by Thermo Fisher Scientific (1 mentions)
Chicken anti beta galactosidase, by Abcam (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
9 protocols using vectashield h 100
1
Quantification of DNA Damage in U87 Cells
2
Cell Preparation for Microscopic Analysis
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Cells were seeded
(2 × 105 cells/well) in 6-wells and left to recover
for 24 h. Then, indicted treatment was added for 24 h after which
cells were collected by trypsinization and transferred to microscopic
slides by centrifugation with the cytocentrifuge Thermo Scientific
Cytospin 4 (400 rpm, 5 min). After drying, cells were fixed on −20
°C with a precooled methanol/aceton (1:1) mixture for 10 min
and dyed with DAPI (1 μg/mL) for another 10 min. For microscopy,
cells were mounted with Vectashield (H-100, Vector Laboratories, Inc.,
CA, USA).
(2 × 105 cells/well) in 6-wells and left to recover
for 24 h. Then, indicted treatment was added for 24 h after which
cells were collected by trypsinization and transferred to microscopic
slides by centrifugation with the cytocentrifuge Thermo Scientific
Cytospin 4 (400 rpm, 5 min). After drying, cells were fixed on −20
°C with a precooled methanol/aceton (1:1) mixture for 10 min
and dyed with DAPI (1 μg/mL) for another 10 min. For microscopy,
cells were mounted with Vectashield (H-100, Vector Laboratories, Inc.,
CA, USA).
3
Chromosome Staining and Imaging Protocol
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CPD staining followed the procedure described by She et al. (2006) (link). Briefly, chromosome preparations were treated with RNase A and pepsin and then stained with a mixture of 0.6 µg·ml-1PI and 3 µg·ml-1DAPI in a 30% (v/v) solution of Vectashield H100 (Vector Laboratories, Burlingame, US) for at least 30 min in the dark at room temperature. Slides were examined under an Olympus BX60 epifluorescence microscope. Separate images from UV and green filters were captured using a cooled CCD camera (CoolSNAP EZ; Photometrics, Tucson, US) controlled using METAMORPH software (Molecular Devices, California, US). DAPI and PI grey scale images of the same plate were merged to produce a CPD image. Final images were optimized for contrast and brightness using ADOBE PHOTOSHOP version 8.01.
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4
Combined PI and DAPI Staining of Chromosomes
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CPD staining was performed following the procedure indicated by She et al. (2006) (link). In brief, the chromosome preparations were sequentially treated with RNase A and pepsin and then stained with a mixture of 0.6 µg·mL-1 PI and 3 µg·mL-1DAPI in a 30% (v/v) solution of Vectashield H100 (Vector Laboratories, Burlingame, US) for more than 30 min. Chromosome spreads were observed using an Olympus BX60 epifluorescence microscope with UV and green excitation filters. Images were captured and merged using a cooled CCD camera (CoolSNAP EZ; Photometrics, Tucson, US) controlled by METAMORPH software (Molecular Devices, California, US).
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5
Mitotic Nuclei Quantification Protocol
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In order to detect mitotic nuclei, fixed cells were stained with DAPI. SW480 and SW480/Coti cells were seeded at a density of 2 × 105 cells per well in 6-well plates and left to recover for 24 h. This was followed by drug treatment at the indicated concentrations for 24 h. Then, cells were collected and spun on slides by centrifugation with a Thermo Scientific Cytospin 4 (400 rpm, 5 min at 4 °C). The slides were dried, and the cells were fixed with a methanol/acetone (1:1) mixture at −20 °C and stained with a solution containing DAPI (1 µg/mL). A final washing step with phosphate-buffered saline (PBS) was performed, followed by covering the slides with vectashield (H-100, Vector Laboratories, Inc., Newark, CA, USA) and coverslips. Mitotic nuclei were counted with Image J in three different areas per slide from three independent experiments.
6
Acetylcholinesterase Immunofluorescence in Brain Tissue
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Brain tissue slices were fixed in 4% paraformaldehyde for 15 min, air-dried, and washed with PBS. Nonspecific proteins were blocked in the blocking serum, consisting of 3% normal donkey serum and 0.3% Triton X-100 in PBS, and incubated at room temperature for 30 min. After blocking, the slices were incubated with primary anti-AChE (Abcam PLC, Waltham, MA, USA). The sections were kept in a humidified box at 4 °C overnight. Tissues were washed three times with PBS and then were incubated for 1 h with donkey antibody-conjugated secondary antibodies conjugated with fluorescent dyes. Excess secondary antibodies were removed by washing three times in PBS and once with deionized H2O. Tissue slides were counter-stained with nuclear staining dye (DRAQ5) and mounted with a mounting medium (Vectashield, H-100, Vector Laboratories, Burlingame, CA, USA). Photomicrographs were obtained using a Leica confocal fluorescence microscope (Leica Microsystems Inc., Bannockburn, IL, USA).
7
COTI-2 Cytotoxicity Assay and ABCC1 Localization
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Cells were seeded 2 ×
204/well in 24-well plates and left to recover for 24 h.
Then, cells
were treated with COTI-2 with indicated concentrations. After 24 h,
microscopy images were taken with a Nikon eclipse Ti-e fluorescence
microscope with differential interference contrast and a sCMOS pco.edge
camera. Images were processed and analyzed by ImageJ. Cells with vacuoles
were counted and given in percent of all attached cells. For immunofluorescence
imaging of ABCC1, cells were transferred to microscopy slides using
a cytocentrifuge (Cytospin 4, Thermo Scientific, USA) at 400 rpm for
5 min. After drying, the cells were fixed with 4% PFA for 15 min at
room temperature and blocked with 20% fetal calf serum in PBS for
1 h. Cells were stained with primary anti-ABCC1 monoclonal mouse
MON9017 (Synbio) (1:50 in 1% bovine serum albumin in PBS) and secondary
anti-mouse conjugated to AlexaFluor488 (Thermo Fisher, 1:200 in 1%
bovine serum albumin in PBS). After washing, counterstain was performed
with DAPI (1 μg/mL) and wheat germ agglutinin (WGA) conjugated
to Rhodamine (10 μg/mL, Vector Laboratories, USA) for 10 min.
Vectashield (H-100, Vector Laboratories, Inc., CA, USA) was used for
mounting, and cells were imaged at the confocal Zeiss LSM 700 Olympus
microscope (Carl Zeiss AG, Oberkochen, Germany) and processed with
ImageJ.
204/well in 24-well plates and left to recover for 24 h.
Then, cells
were treated with COTI-2 with indicated concentrations. After 24 h,
microscopy images were taken with a Nikon eclipse Ti-e fluorescence
microscope with differential interference contrast and a sCMOS pco.edge
camera. Images were processed and analyzed by ImageJ. Cells with vacuoles
were counted and given in percent of all attached cells. For immunofluorescence
imaging of ABCC1, cells were transferred to microscopy slides using
a cytocentrifuge (Cytospin 4, Thermo Scientific, USA) at 400 rpm for
5 min. After drying, the cells were fixed with 4% PFA for 15 min at
room temperature and blocked with 20% fetal calf serum in PBS for
1 h. Cells were stained with primary anti-ABCC1 monoclonal mouse
MON9017 (Synbio) (1:50 in 1% bovine serum albumin in PBS) and secondary
anti-mouse conjugated to AlexaFluor488 (Thermo Fisher, 1:200 in 1%
bovine serum albumin in PBS). After washing, counterstain was performed
with DAPI (1 μg/mL) and wheat germ agglutinin (WGA) conjugated
to Rhodamine (10 μg/mL, Vector Laboratories, USA) for 10 min.
Vectashield (H-100, Vector Laboratories, Inc., CA, USA) was used for
mounting, and cells were imaged at the confocal Zeiss LSM 700 Olympus
microscope (Carl Zeiss AG, Oberkochen, Germany) and processed with
ImageJ.
8
Gut Morphology and Stem Cell Staining
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Adult guts were dissected in PBS and fixed twice during 20 min in fresh 4% paraformaldehyde diluted in PBS (18814-10, Polysciences) with a 10 min wash in PBT 0,1% (PBS + Triton X100 Sigma Aldrich) in between, apart for fig. 2B-F, where guts were fixed once. Samples were then washed 5 min three times in PBT 0.1% and permeabilised for 30 min in PBT 1%. All samples were then incubated 30min at room temperature in blocking buffer (PBS + 1% BSA, A2153-10G, Sigma) followed with primary antibodies incubation in blocking buffer overnight at 4°C. Samples were then washed 3 times 15min in PBT 0.1%, subjected to secondary antibody staining in blocking buffer for 2 hours at room temperature followed by 3 washings in PBT 0.1%. Guts were mounted in Vectashield (H-100, Vector Laboratories) between coverslip and glass slide. The following primary antibodies were used: chicken anti-GFP, 1/1000 (Abcam 13970); mouse anti-Delta, 1/1000 (C594.9B-c, DSHB); chicken anti-Beta galactosidase, 1/1000 (Abcam 9361); mouse anti-Armadillo (b-Catenin), 1/50 (N2 7A1-c, DSHB), mouse anti-V5, 1/400 (R96025, ThermoFisher), rat anti-a--Catenin, 1/20 (DSHB D-CAT1), rabbit anti-PH3(Ser10), 1/500 (9701 CST); mouse anti-Prospero, 1/50 (DSHB, MR1a). Alexa-488, 555 and 647 conjugated secondary goat antibodies or Phalloidin were used (Molecular probes) and nuclei were counterstained with DAPI.
9
Immunostaining of Adult Fly Brains
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The whole brains of adult flies were dissected in ice-cold haemolymph-like saline (HL3) solution (Stewart et al., 1994) , fixed for 20 min in 4% paraformaldehyde (PFA) in PBS and permeabilized in PBS-T (1% Triton TM X-100) for 20 min at RT. Samples were blocked in 10% normal goat serum (NGS) in PBS-T (0.3% Triton TM X-100) for at least two hours. Brains were incubated with the indicated primary antibody (1:500) in brain staining buffer (5% NGS, 0.1% NaN 3 in PBS-T (0.3% Triton TM X-100)) for 48 hours at 4 C. Subsequently, brains were washed in PBS-T (0.3% Triton TM X-100) for 24 hours at 4 C with multiple buffer exchanges. Next, samples were incubated with appropriate secondary antibody in brain staining buffer for 24 hours at 4 C, washed six times for 30 min in PBS-T (0.3% Triton TM X-100) at RT and stored in VectaShield H-100 (Vector Laboratories) at least for one day at À20 C. Brains were mounted and imaged using the Leica TCS SP8 Confocal Microscope. Images were analyzed using Fiji.
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