RPE1 cells cultured on coverslip were washed with PBS and fixed in 4% PFA at RT for 30 min, permeabilized in 0.1% Triton X-100 (T8787, Sigma, Saint Louis, MO, USA)/PBS for 15 min, washed with PBS, and then blocked in 5% FBS/PBS for 1 h at RT. Samples were then incubated with a primary antibody (diluted in blocking solution) at 4 °C overnight, washed several times with the blocking solution, and then incubated with a secondary antibody for 2 h at RT. After several washes, DNA was counterstained with DAPI (D9564, Sigma, Saint Louis, MO, USA). Samples were then mounted with mounting solution (P36934, Life Technologies, Carlsbad, CA, USA) and imaged using the Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany). Primary and secondary antibodies used in this study were listed in Supplementary Table S1 .
P36934
Manufactured by Thermo Fisher Scientific
Sourced in United States
The P36934 is a laboratory equipment product offered by Thermo Fisher Scientific. It is designed to perform a core function within the laboratory setting. However, a detailed and unbiased description of its specific features and capabilities is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Axiovert 200m, by Zeiss (4 mentions)
D1306, by Thermo Fisher Scientific (3 mentions)
T8787, by Merck Group (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Dapi d9564, by Merck Group (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Sigmaplot, by Grafiti LLC (1 mentions)
Ab111346, by Abcam (1 mentions)
Lsm 5 exciter, by Zeiss (1 mentions)
Ab3274, by Merck Group (1 mentions)
P2031, by Biosesang (1 mentions)
Alexa fluor 488 goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
Lsm 980 airyscan microscope, by Zeiss (1 mentions)
Tissue tek, by Sakura Finetek (1 mentions)
Sc 28385, by Santa Cruz Biotechnology (1 mentions)
Bz x700, by Keyence (1 mentions)
Chamber slides, by NEST Biotechnology (1 mentions)
Ab7028, by Abcam (1 mentions)
A21207, by Thermo Fisher Scientific (1 mentions)
14 protocols using p36934
1
Immunofluorescence Staining of RPE1 Cells
2
Proximity Ligation Assay for Cell Analysis
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Proximity Ligation Assay was performed without deviation from manufacturer’s instructions (DUO92008). Coverslips were washed in a 0.5mL volume and reactions were performed by inverting the coverslip onto a 35μL drop on parafilm. Following the proximity ligation reaction, cells were stained with DAPI (0.2μg/mL) for 3 minutes followed by one wash in PBS and one water wash. The cells were then inverted and mounted on glass coverslips with 15μL of prolong gold mounting media (LifeTech, P36934) & were cured overnight in the dark at room temperature.
All immunofluorescence images were taken on a Zeiss Laser-Scanning Confocal Microscope 980 and analyzed with CellProfiler. All cells within a 63x image was analyzed (~30cells/condition/biological replicate). Statistical significance was assigned using t-test or one-way ANOVA with multiple comparisons.
All immunofluorescence images were taken on a Zeiss Laser-Scanning Confocal Microscope 980 and analyzed with CellProfiler. All cells within a 63x image was analyzed (~30cells/condition/biological replicate). Statistical significance was assigned using t-test or one-way ANOVA with multiple comparisons.
3
Immunofluorescence Analysis of Talpid3 in RPE1 Cells
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RPE1 cells were grown on a coverslip and fixed with cold methanol for 10 min or 4% PFA form 15 min. The cells were incubated with 3% BSA-0.3% PBST (Triton X-100) for 20 min. Primary and secondary antibodies in the blocking solution were placed on the cells for 2 hrs at RT Antibodies were diluted in 0.1% PBS with Triton X-100 with 3% BSA. The cells were mounted onto a slide glass with mounting solution (P36934; Life Technologies) and imaged using a microscope (63× or 100×, NA 1.4; Axiovert 200M, Carl Zeiss, Germany) equipped with a cooled CCD (Retiga 2000R; QImaging, Surrey, Canada) and MetaMorph . The images were obtained as z projections (0.3 µm interval) and analyzed by ImageJ (NIH, http:// rsbweb.nih.gov/ij/ ) and SigmaPlot (Systat Software, San Jose, CA). The region of interest was defined by drawing a circle including the centrosome. Background values were measured from the same-sized circle as a circle including the centrosome in an adjacent region. Talpid3 staining was analyzed in G1 phase cells (serum starved for 24 or 48 hr) to achieve uniformity and to avoid oscillations in abundance during the cell cycle.
4
Immunofluorescence Microscopy of Aquaporin-2
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Cells were grown on semipermeable filter supports in a transwell chamber (0.4 μm pore size, Transwell Permeable Supports, 3460, Corning, NY, USA) and treated with vehicle or 10−9 M dDAVP applied to the basolateral side for 30 min without a period of dDAVP-mediated AQP2 induction. Cells were washed twice in PBS and then fixed with 4% paraformaldehyde (P2031, Biosesang, Seoul, Korea) for 30 min at room temperature. Cells were washed twice in PBS and permeabilized with 0.3% Triton X-100 (T8787, Sigma, St. Louis, MO, USA) for 15 min at room temperature. Following permeabilization, cells were incubated with anti-AQP2 antibody (1:200, AB3274, Millipore, Burlington, MA, USA) and anti-phosphorylated AQP2 at serine 256 (1:100, ab111346, Abcam, Cambridge, UK) in PBS at 4 ℃ overnight. Cells were washed and incubated with goat-anti-rabbit IgG Alexa Fluor 488 secondary antibody (A11008, Invitrogen, Carlsbad, CA, USA) for 2 h at room temperature. The nuclei were stained with DAPI (D1306, Invitrogen, Carlsbad, CA, USA) for 30 min at room temperature and cells were mounted with an antifading reagent (P36934, Invitrogen, Carlsbad, CA, USA). Immunofluorescence microscopy was carried out using a laser scanning confocal microscope (Zeiss LSM 5 EXCITER, Jena, Germany).
5
Tubulin Polymerization Assay with Compounds
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TAMRA-Tubulin (TL590M, Cytoskeleton) diluted in GTB buffer (PEM, 10% glycerol, 1 mM GTP) was mixed with unlabeled α,β-tubulin (T240, Cytoskeleton) at a ratio of 1:3 (v/v) to a concentration of 4.0 mg/mL. Tubulin polymerization was performed at 37°C for 45 min in the presence of 5% glycerol and 20 μM of test compounds (0.08% DMSO, 26Z , CA-4, and paclitaxel), and it was initiated with the addition of 1 mM GTP. Samples were then centrifuged for 20 min at 30,000× g. The pellets were diluted in 6 µL PEM buffer, containing 1 mM GTP and 0.5% glutaraldehyde, mixed with anti-fade mounting media (P36934, Invitrogen, Eugene, USA), and examined under a confocal LSM 980 Airyscan microscope (Carl Zeiss AG, Oberkochen, Germany).
6
Cochlear Immune Cell Visualization
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The cochlea was isolated from the skull, and the stapes and partial cochlear apical bones were removed to allow a fixative solution to flow into the cochlea. The isolated cochlea was fixed in 4% paraformaldehyde overnight at 4 ℃ and decalcified with 0.2 M ethylenediaminetetraacetic (EDTA) in PBS for 2 days. The cochlear tissue was dehydrated with 30% sucrose in PBS overnight at 4 ℃ and embedded in Optimal Cutting Temperature (OCT) compound (Tissue-Tek®, Sakura Finetek, Torrance, CA, USA). The cochlear tissues were sectioned into 12-μm-thick slices using a cryostat. Tissue sections were permeabilized with Triton-X (0.3%, 30 min) and blocked with BSA (4%, 1 h) at room temperature (RT). The sections were then incubated with anti-F4/80 antibody in 1% BSA overnight at 4 ℃, followed by secondary antibody incubation (Alexa 488 or Cy3-labeled anti-Rat). The sections were further incubated with DAPI (D1306, Invitrogen, Waltham, MA, USA). Finally, cover slips were placed on slides after adding the mounting media (P36934, Invitrogen). Images were captured using a confocal microscope (LSM 780, Zeiss, Oberkochen, Germany) and the ZEN 2011 software. F4/80 positive cells were quantified using the Image J software.
7
Fluorescence Imaging of ANXA2, γ-H2AX, and COL6
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Fluorescence cell imaging procedures were performed as follows7 (link). Cells were fixed with 4% paraformaldehyde at room temperature for 20 min, and were permeabilized with 0.1% Triton X-100/10% FBS/PBS for 1 h. To detect ANXA2, γ-H2AX, or COL6, cells were stained using a monoclonal mouse anti-human ANXA2 antibody (200 µg/mL, sc-28385, Santa Cruz Biotechnology, Dallas, TX) at a dilution of 1:50, a mouse monoclonal anti-human phospho-histone H2AX (Ser139) antibody at a dilution of 1:250, and a polyclonal rabbit anti-human COL6 antibody at a dilution of 1:2500, respectively. After staining with each primary antibody, cells were stained using the following secondary antibodies: the goat anti-mouse IgG labeled with Alexa Fluor 594 and goat anti-rabbit IgG labeled with Alexa Fluor 488 at a dilution of 1:1000. Nuclei were stained with 6-diamidino-2-phenylindole (DAPI) (P36934, Invitrogen). Fluorescence images were detected under fluorescent microscopes (BZX700, Keyence, Osaka, Japan, BX-51, Olympus, or IX71, Olympus). The number of cells that were positive for γ-H2AX in the nucleus or COL6 in the cytoplasm was determined using Win ROOF 2015 software. The signal intensity of ANXA2 (Fig. 5 b) was measured by Image J31 (link).
8
Quantifying Cell Proliferation via BrdU Staining
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Cells were seeded on chamber slides (NEST, Wuxi, China), cocultured with BrdU for 5 hours (60 μg/mL), washed with phosphate-buffered saline (PBS), and fixed with methanol for 5 minutes at room temperature in the dark. Slides were washed with PBS and incubated with PBS containing 2 mol/L HCl and 0.1% Tween 20 to break the double-stranded DNA. Next, 0.1 mol/L sodium borate was used to stop the reaction, and slides were washed in PBS, followed by incubation in 0.1% Tween 20 mixed with 10% goat serum to increase the permeability of the nuclear membrane and to block nonspecific antibody binding. The slides were incubated with anti-BrdU antibody overnight at 4°C and then with Alexa Fluor 594 goat anti-rat IgG (1:500) secondary antibody at room temperature for 1 hour. The slides then were incubated with 4′,6-diamidino-2-phenylindole at room temperature for 15 minutes to visualize DNA. Finally, the slides were fixed with mounting medium (p36934; Invitrogen).
9
Immunolocalization of Pluripotency and Lineage Markers
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The embryos were fixed with 4% paraformaldehyde (PFA), washed with 0.1% polyvinyl alcohol in PBS (PVA-PBS) and treated with 0.5% Triton X-100 for 30 min. After incubation with 0.25% Tween-20 in PBS for 30 min, the embryos were blocked with 3% BSA in PVA-PBS for 2 h and then washed with 0.1% PVA-PBS at room temperature. The embryos were incubated at 4 8C overnight with the following primary antibodies: anti-OCT4 (POU5F1) (sc-9081; Santa Cruz Biotechnology, 1:1000 dilution) and anti-CDX2 (MU392A-UC; BioGenex, San Ramon, CA, USA, 1:1000 dilution). After incubation with donkey anti-rabbit-594 (A21207, Invitrogen, 1:500 dilution) and goat anti-mouse 488 (A11029, Invitrogen, 1:500 dilution) secondary antibodies, the embryos were counterstained with DAPI and mounted with a mounting medium.
For immunocytochemistry analysis, the cultured MEFs were fixed in 4% PFA, washed with 0.1% Tween-20 in PBS (PBST) and treated with 0.25% Triton X-100 in PBS. After washing with PBST, the cells were blocked with 2% BSA in PBST for 1 h at room temperature and incubated at 4 8C overnight with an anti-HDAC1 primary antibody (ab7028; Abcam, Cambridge, MA, USA, 1:5000 dilution). The cells were then washed in PBST, incubated with secondary antibodies, stained with Hoechst33342 (Sigma), mounted with a mounting medium (P36934, Invitrogen) and analyzed using a Leica TCS SP5 II confocal microscope.
For immunocytochemistry analysis, the cultured MEFs were fixed in 4% PFA, washed with 0.1% Tween-20 in PBS (PBST) and treated with 0.25% Triton X-100 in PBS. After washing with PBST, the cells were blocked with 2% BSA in PBST for 1 h at room temperature and incubated at 4 8C overnight with an anti-HDAC1 primary antibody (ab7028; Abcam, Cambridge, MA, USA, 1:5000 dilution). The cells were then washed in PBST, incubated with secondary antibodies, stained with Hoechst33342 (Sigma), mounted with a mounting medium (P36934, Invitrogen) and analyzed using a Leica TCS SP5 II confocal microscope.
10
Immunofluorescence Staining of Mouse Brain
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Immunofluorescence (IF) staining of mouse brain sections was performed as previously described (31 (link)). Slides were washed 3 times for 15min with PBS to remove residual OCT. The sections were then incubated in the blocking solution (PBS containing 10% donkey serum (cat no: S30-100ml, Millipore Sigma), 2% BSA (Fisher Scientific, BP1600-100) and 0.3% Triton X-100 (Fisher Scientific, BP151-100) for 2h at room temperature (RT). Sections were then transferred to blocking solution containing the primary antibodies (NBR1 (proteintech, 16004-1-AP), IBA1 (Novus Biologicals, NB100-1028), and incubated overnight at 4°C. After that, sections were washed with PBS 3 times for 15min each. Then, they were incubated with the blocking solution containing the secondary antibody Donkey anti-Rabbit IgG (ThermoFisher Scientific, A32790), Donkey anti-Goat IgG (cat. no: A-11058, ThermoFisher Scientific) for 2h at RT. DAPI (Fisher Scientific, D1306) was added on the top of the antibody solution in the last 15min of the incubation period at a final concentration (5ug/ml). Finally, sections were washed with PBS 3 times for 15min. Antifade mounting media (ThermoFisher Scientific, P36934) was added before cover-slipped.
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