CGH and FISH with microsatellite motifs probes were conducted using methods described in our previous study [9] (link), [25] (link). For chromosome probes, we conducted FISH with slight modification. Chromosome probes were labelled by nick translation incorporating SpectrumGreen-dUTP (Abbott, North Chicago, Illinois, USA) or SpectrumOrange-dUTP (Abbott). Each labelled probe was precipitated with 20 µg glycogen as carrier, and dissolved in 15 µl hybridization buffer. The hybridization mixture was placed on a chromosome slide and sealed with a coverslip and rubber cement. Probe DNA and chromosome DNA were denatured by heating the slide on a heat plate at 68.5°C for 5 min. The slides were hybridized overnight in a humid chamber at 37°C. Hybridization was carried out for 2 days in cross-species chromosome painting. The slides were then washed by the following series: 0.4×SSC, 0.3% IGEPAL (Sigma-Aldrich) at 55°C for 2 min followed by 2×SSC, 0.1% IGEPAL at room temperature for 1 min. The slides were dehydrated by ethanol series and air-dried and then counterstained using 20 µg/ml DAPI (4′,6-diamidino-2-phenylindole), 2×SSC and mounted with anti-fade medium, Vectashield (Vector Laboratories, Burlingame, California, USA).
Igepal
Manufactured by Merck Group
Sourced in United States, United Kingdom, Canada, Germany, Japan, Italy, Switzerland, Australia
IGEPAL is a nonionic detergent product manufactured by Merck Group. It is primarily used as a surfactant and emulsifier in various laboratory applications. The core function of IGEPAL is to aid in the solubilization, dispersal, and stabilization of materials in aqueous solutions.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (16 mentions)
Rnaseout, by Thermo Fisher Scientific (10 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (9 mentions)
Bovine serum albumin (bsa), by Merck Group (8 mentions)
Protease inhibitor cocktail, by Roche (7 mentions)
Triton x 100, by Merck Group (7 mentions)
Indomethacin, by Merck Group (6 mentions)
Paraformaldehyde (pfa), by Merck Group (6 mentions)
Sodium chloride (nacl), by Merck Group (6 mentions)
Lsm 510 meta, by Zeiss (6 mentions)
Puromycin, by Merck Group (6 mentions)
D luciferin, by GoldBio (5 mentions)
Coenzyme a, by Merck Group (5 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (5 mentions)
Insulin, by Merck Group (5 mentions)
Dexamethasone (dex), by Merck Group (5 mentions)
Infinite m200 plate reader, by Tecan (5 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (5 mentions)
Complete protease inhibitor cocktail, by Roche (5 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (5 mentions)
238 protocols using igepal
1
Fluorescent In Situ Hybridization with Microsatellite Probes
2
Trypsinization and Apoptosis Measurement of Neurospheres
3
Extraction of Aggregated Proteins from Tissue
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100 mg of frozen cortical tissue was homogenised in ~1:10 (w/v) Igepal (Sigma, Dorset, UK) based lysis buffer (in mM: 20 HEPES, 150 NaCl, 0.1 EDTA, 1 % Igepal: pH = 7.6). All buffers were supplemented with complete protease inhibitors (Roche) and PhosStop tablets (Roche). The use of the non-ionic, non-denaturing Igepal, which is chemically indistinguishable from the widely used ‘Nonidet P-40’, ensured adequate lysis of plasma, endoplasmic and Golgi but not nuclear membranes, and prevented aggressive solubilisation of large aggregates [33 (link)]. Following manual homogenisation, samples were spun (13,000g, 4 °C, 20 min), supernatants separated from pellets, aliquoted and stored at −80 °C. For the determination of aggregated non-soluble protein pathology, Igepal derived pellets were re-suspended in excess Igepal buffer (1 ml) homogenised via repeated aspiration with a 1 ml pipette tip, briefly vortexed and spun (13,000g, 4 °C, 20 min). Following removal of the supernatants, the process was repeated. Resulting pellets were subsequently re-suspended in 1:1 (w/v) 70 % formic acid, incubated overnight at 4 °C with continuous agitation before a final spin (18,000g, 4 °C, 20 min) to yield the collected supernatant, which was stored at −80 °C until use.
4
Co-immunoprecipitation Assay Protocol
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Co-immunoprecipitation assays were performed as described previously (15 (link)). Total soluble protein was extracted as described above from leaves of N. benthamiana 3 days after agroinfiltration using the GTEN buffer [10% glycerol, 25 mM tris-HCl (pH 7.5), 1 mM EDTA, and 150 mM NaCl] supplemented with 2% (w/v) polyvinylpolypyrrolidone (PVPP), 10 mM DTT, and 1× protease inhibitor cocktail (Sigma-Aldrich), as well as 0.3% IGEPAL (Sigma-Aldrich). OD600 used can be found in table S2. Protein extracts were filtered using Minisart 0.45-μm filters (Sartorius Stedim Biotech, Goettingen, Germany). Part of the extract was set aside before immunoprecipitation and used for SDS-PAGE as described above. These were the inputs. A total of 1.4 ml of the remaining filtered total protein extract was mixed with 30 μl of anti–c-Myc agarose beads (A7470, Sigma-Aldrich) and incubated end over end for 90 min at 4°C. Beads were washed five times with immunoprecipitation wash buffer [GTEN extraction buffer with 0.3% (v/v) IGEPAL (Sigma-Aldrich)] and resuspended in 60 μl of SDS loading dye. Proteins were eluted from beads by heating for 10 min at 72°C. Immunoprecipitated samples were used for SDS-PAGE and immunoblotted as described above and compared to the inputs.
5
Fluorescence In Situ Hybridization (FISH) Analysis
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FISH was performed using a Texas red‐conjugated chromosome 17 probe (Empire Genomics, CHR17‐10‐RE) following the manufacturer's instructions. Briefly, the slides were fixed for 45 min using Histochoice (AMRESCO, Solon, OH) and then dehydrated in ethanol. The probe mixture (2 µl of probe and 8 μl of buffer) was added onto the section and covered with a cover glass, which was further sealed with rubber cement. The slide set was first placed in 83°C warmer for 3 m for probe denaturation, then was placed in a humidified chamber and incubated at 37°C for 16 h. After incubation, the slide was removed from the humidified chamber, and the cover glass was removed. The slide was washed in wash solution 1 (WS1, 0.3% Igepal, Sigma CA‐630) at 73°C for 2 m, then washed in WS2 (0.1% Igepal) at room temperature for 2 m. After the slide was air dried in dark, mounting medium containing DAPI was added onto the section. The slides were examined under a Nikon Eclipse T300 fluorescence microscope.
6
Fluorescent In Situ Hybridization Protocol
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To hybridize the probe, we followed the manufacturer's recommendations. In brief, we resuspended the probe to a concentration of 100 ng/µL. We used 200 ng per slide diluted into 38 µL hybridization buffer (BioCare Medical, Pacheco, CA), and then coverslips were sealed with rubber cement. The slides were denatured at 68 °C for 5 min and then incubated at 37 °C for 24–48 h. Following incubation, the coverslips were removed, and the slides were washed with 0.4× SSC: 3 M NaCl, 0.3 M sodium citrate, pH 7, and 0.3% (v/v) IGEPAL (Sigma-Aldrich) at 60 °C for 2 min, followed by a second wash at room temperature with 2× SSC: 3 M NaCl, 0.3% M sodium citrate, pH 7, and 0.1% (v/v) IGEPAL (Sigma-Aldrich) for 1 min. The slides were then desiccated with an ethanol wash series of 70%, 90%, and 100% (v/v) for 1 min each and allowed to dry completely. Once dry, the slides were stained with Vectashield antifade mounting medium with DAPI (Vector Laboratories) and viewed and photographed with a Leica Microsystems Thunder Imaging system. Karyotype images were constructed from metaphase chromosomes using Adobe Photoshop 2021.
7
FISH Analysis for BCL6, MYC, and BCL2
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Sample processing, hybridization and analysis were performed as previously described [22 (link)] using dual color, break apart probes flanking the BCL6 (3q27), MYC (8q24) and BCL2 (18q21) genes (Zytovision, Bremerhaven, Germany). Briefly, slides were deparaffinized, re-hydrated in 2× SSC, and placed in a solution of 1 mol/L sodium sulfocyanate at 80 °C for 10 min (Merck, Darmstadt, Germany). Following tissue digestion with 6 mg/mL pepsin (Sigma-Aldrich, Steinheim, Germany) for 6 min at 37 °C, slides were rinsed in 2× SSC and dehydrated in a series of ethanol. Ten µL of probe was applied to each slide and codenatured at 80 °C for 8 min. Hybridization took place for 18 h at 37 °C followed by post hybridization washes in 2× SSC/0.5% Igepal (Sigma-Aldrich) at 74 °C for 5 min and 2× SSC/0.1% Igepal at room temperature for 3 min. Slides were counterstained with DAPI and 100 morphologically intact, non-overlapping nuclei were scored.
8
Co-immunoprecipitation Protocol for Yeast
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Yeast strains used for co-immunoprecipitation experiments are listed in Table S4. Cells were grown as 50-ml cell cultures to mid log phase (OD=0.6), collected, and lysed using buffer A [20 mM Hepes pH 7.6, 20% glycerol, 1 mM DTT, 1 mM EDTA, 125 mM potassium acetate, 1% NP-40 (IGEPAL, Sigma), 1 mM phenylmethylsulfonyl fluoride, 1X protease inhibitor cocktail (Sigma) and 1X phosphatase inhibitor cocktail (Sigma)] (Moqtaderi et al., 1996) . For the FACT-histone co-IP experiments, lysates from strains FY3303-3306 were prepared using buffer B for lysis and coimmunoprecipitation [100 mM Hepes pH 7.9, 20% glycerol, 1 mM EDTA, 25 mM magnesium acetate, 0.4% NP-40 (IGEPAL, Sigma), 1 mM phenylmethylsulfonyl fluoride, 1X protease inhibitor cocktail (Sigma)]. Cell lysates were diluted with the lysis buffer to achieve equal protein concentrations (about 1 mg total protein per IP) and were then used for pull-downs with the respective antibody-conjugated beads: either the anti-FLAG M2-FLAG affinity gel (Sigma) (20 µl per IP) or anti-V5-conjugated magnetic beads (MBL International Corporation) (30 µl per IP) as previously described (Reim et al., 2020) . The eluates were analyzed using western blotting. The antibodies used are listed in the Key Resource Table.
9
Preparation and Validation of Sex Chromosome Probes
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Animal collection, microdissection, preparation of sex chromosome specific probes and validation of probes were described in our previous studies 18, 19, 20 . Briefly, we labelled sex chromosome probes by nick translation incorporating SpectrumGreen-dUTP (Abbott, North Chicago, Illinois, USA) or SpectrumOrange-dUTP (Abbott) and precipitated with 20 µg glycogen. After decantation, labeled probe pellets were resuspended in a 15 µl hybridization buffer. The resuspended probe mixture was hybridized with a drop of metaphase chromosome suspension fixed on a glass slide, covered with coverslips, and sealed with rubber cement. The slide was then denatured on a hot plate at 68.5°C for 5 min and was hybridized overnight in a humid chamber at 37°C for two days. The slides were then washed first with 0.4×SSC, 0.3% IGEPAL (Sigma-Aldrich) at 55°C for 2 min followed by 2×SSC, 0.1% IGEPAL for 1 min at room temperature. The slides were dehydrated by ethanol series and air-dried and then mounted with anti-fade medium Vectashield (Vector Laboratories, Burlingame, California, USA) containing 20 µg/ml DAPI (4′,6-diamidino-2-phenylindole.).
10
Highly Crosslinked H3K27ac ChIP-qPCR Protocol
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H3K27ac chromatin immunoprecipitation was performed as described68 . The CBP and p300 ChIP experiments required the following adjustments: minced hippocampal tissue was fixed in 1% PFA for 30 min at 37 °C (as suggested in ref. 69 (link)), which allowed for crosslinking of the KAT3 cofactors to the DNA-binding proteins and to the DNA itself. Because KAT3 proteins are almost completely degraded after a few rounds of sonication in 1% sodium dodecyl sulfate (SDS), the sonication buffer was changed to one containing 0.1% SDS, 1% IGEPAL (Sigma-Aldrich), and 0.5% sodium deoxycholate. Ten additional sonication cycles of 30″ on/30″ off were added to fragment the highly fixed DNA. ChIP-qPCR assays were performed in a QuantStudio 3 unit using the primer pairs indicated in Supplementary Table 2 .
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