Embryos were isolated from superovulated female mice (Myo10flox/flox; Cre+ and Myo10flox/flox; Cre− as Myo10−/− full mice, and Myo10wt/wt; Cre+ and Myo10wt/wt; Cre− as control mice) mated with male mice (Myo10wt/wt; Cre−). Superovulation of female mice was induced by intraperitoneal injection of 5 IU pregnant mare’s serum gonadotropin (Ceva, Syncro-part), followed by intraperitoneal injection of 5 IU human chorionic gonadotropin (MSD Animal Health, Chorulon) 44–48 h later. Embryos were recovered at E0.5 from plugged females by opening the ampulla followed by a brief treatment with 37°C 0.3 mg/ml hyaluronidase (H4272-30MG; Sigma-Aldrich) and washing in 37°C FHM. When ampulla was not present, embryos were recovered by flushing oviducts with 37°C FHM (MR-122-D; Millipore) using a modified syringe (1400 LL 23; Acufirm). Embryos were handled using an aspirator tube (A5177-5EA; Sigma-Aldrich) equipped with a glass pipette pulled from glass micropipettes (Blaubrand intraMark or Warner Instruments). Embryos were placed in KSOM (MR-107-D; Millipore) supplemented with 0.1% BSA (A3311; Sigma-Aldrich) in 10 ml droplets covered in mineral oil (M8410; Sigma-Aldrich). Embryos were cultured in an incubator under a humidified atmosphere supplemented with 5% CO2 at 37°C for 5 d. Embryos were scored for survival and embryonic stage from E0.5 to E4.5.
A3311
Manufactured by Merck Group
Sourced in United States
The A3311 is a piece of laboratory equipment designed for general laboratory use. It is a compact and versatile instrument that can perform a variety of tasks. The core function of the A3311 is to provide a controlled environment for various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Human chorionic gonadotropin (hcg), by MSD (1 mentions)
M8410, by Merck Group (1 mentions)
A5177 5ea, by Merck Group (1 mentions)
Mr 107 d, by Merck Group (1 mentions)
Human chorionic gonadotropin (hcg), by MSD animal health (1 mentions)
880 confocal microscope, by Zeiss (1 mentions)
Sc 32293, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 647 donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
P36962, by Thermo Fisher Scientific (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Ab3583, by Abcam (1 mentions)
Western blotting detection reagent, by GE Healthcare (1 mentions)
Imagequant 350, by GE Healthcare (1 mentions)
Wl01114, by Wanlei (1 mentions)
Cooled ccd camera, by Nikon (1 mentions)
Iseq00010, by Merck Group (1 mentions)
Human chorionic gonadotropin (hcg), by Merck Group (1 mentions)
Am9261, by Thermo Fisher Scientific (1 mentions)
Intesticulttm organoid growth medium, by STEMCELL (1 mentions)
Matrigel matrix, by Corning (1 mentions)
13 protocols using a3311
1
Embryo Isolation and Culture for Myosin-X Knockout Mice
2
Visualizing Microtubule Dynamics with Cytochalasin D
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Autoclaved 13 mm coverslips were placed in wells of a 24-well dish and coated with 0.25 μg/ mL Concanavalin A (L7647, Sigma) for at least 1 h and up to over-night at 37°C. Coverslips were washed twice with PBS before plating 1.5 * 105 cells/well with either DMSO or 2.5 μM CytD. DMSO concentrations were adjusted to 0.125% in both samples. Cells were grown for 5 h before fixing for 10 min in fixation buffer (3% paraformaldehyde, 0.1% Glutaraldehyde, 4% Sucrose, 0.5% Triton-X-100, 0.1 M PIPES pH 7, 2 mM EGTA, 2 mM MgCl2). This and all the following steps were performed at room temperature. Samples were washed 3 times with PBS, permeabilized with 0.1% Triton-X-100 in PBS for 10 min and washed again with PBS. Samples were blocked with 2% BSA (A3311, Sigma) in PBS for 30 – 60 min and then incubated with primary antibody against α-tubulin (sc-32293, Santa Cruz) for 1 h. Samples were washed with PBS and incubated with secondary Alexa Fluor 647 donkey anti-mouse (life technologies) for 1 h. Samples were mounted with mounting media (P36962, Invitrogen) and left to cure over-night before imaging on a Zeiss 880 confocal microscope equipped with a 63x/1.4 NA Oil lens. Z-stacks including the entire volume of the cell (24 slices, 0.63 μm/ pixel) were acquired and summed to generate a projection image showing all microtubules within the cell.
3
Western Blot Analysis of OB and OBR Proteins in Ram Reproductive Tissues
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Western blot was used for analysis of OB and OBR proteins in testis, epididymal tissue, ejaculated and epididymal sperm of rams as described by the method of Zhang (Zhang et al., 2017 (link)). Briefly, tissues or cells were lysed with a lysis buffer (HX1862; Huaxingbo, Hong Kong, China). Equal amounts of protein were resolved using 12% SDS-PAGE gel and transferred to PVDF membranes (ISEQ00010; Millipore, Burlington, MA, USA). After they were blocked with 5% nonfat milk or BSA (A3311; Sigma-Aldrich, St. Louis, MI, USA) at 37 °C for 60 min, the membranes were incubated with primary antibodies against OB (1:1,000, ab3583, Abcam, Cambridge, UK), OBR (1:1,000, 20966-1-AP, Proteintech, Rosemont, IL, USA) and GAPDH ( 1:2,500, WL01114; Wanleibio, Hebei, China) at 4 °C, overnight. The membranes were then washed 3–5 times with TBST buffer (HX1893; Huaxingbo, Hong Kong, China) and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000, 7074; Cell Signaling Technology, Danvers, MA, USA). Western blotting detection reagents (GE Healthcare, Uppsala, Sweden), and then were visualized using a cooled CCD camera (Nikon, Tokyo, Japan) based chemiluminescence detector (GE Healthcare, Uppsala, Sweden). Protein bands were then analyzed by computer software (Image-Quant 350; GE Healthcare, Uppsala, Sweden).
4
Mouse Oocyte Fertilization and Embryo Preparation
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Eight- to ten-week-old ICR female mice (Japan SLC, Hamamatsu, Japan) were superovulated by injection of 5 IU of equine chorionic gonadotropin (eCG; ASUKA Pharmaceutical, Tokyo, Japan), followed by 5 IU of human chorionic gonadotropin (hCG; ASUKA Pharmaceutical) 48 h later. Unfertilized eggs were harvested 14 h after hCG injection and placed in a 90-μl droplet of HTF supplemented with 4 mg/ml BSA (A3311; Sigma-Aldrich, St. Louis, MO, USA) [13 (link)]. Spermatozoa were collected from the cauda epididymis of 11- to 15-week-old ICR male mice (Japan SLC) and cultured for 2 h in 100-μl of HTF supplemented with 4 mg/ml BSA. After preincubation, sperm were introduced into fertilization droplets at a final concentration of 1 × 106 cells/ml. After a 3-h incubation, fertilized 1-cell embryos were collected and washed 3 times in KSOM supplemented with amino acids [14 (link)] and 4 mg/ml BSA and then used for
microinjection [15 (link)].
microinjection [15 (link)].
5
Isolation and Culture of Mouse Intestinal Organoids
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The small intestines of mice were opened longitudinally and washed repeatedly with cold PBS (containing 100 U/mL or 100 μg/mL penicillin/streptomycin) until there were no visible impurities. The tissue was cut into 2–3 mm pieces, which were placed in cold 2.5 mM EDTA (AM9261, Invitrogen) at 4°C for 30 min. After removal of the EDTA medium, the tissue fragments were shaken to release crypts and then passed through a 70 μm cell strainer to remove the remaining villi. Isolated crypts were washed with cold PBS containing 0.1% BSA (A3311, Sigma) and centrifuged (∼290 × g) 10 times for enrichment. The crypts were buried in 30 μL cold Matrigel® matrix (356231, Corning) and 30 μL IntestiCultTM Organoid Growth Medium (06005, STEMCELL) at a density of 200 crypts per well. Fresh medium was replaced every 3 days.
6
Coating PMMA Microbeads with Cdh1 Protein
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PMMA microbeads (Microparticles, PMMA-R-B375) 36 μm in diameter, washed in 0.01% Tween20 (Sigma, P-7949) in DPBS (DPBS-T), were used to establish contact with 8-cell stage blastomeres. To coat microbeads with Cdh1, recombinant mouse Cdh1-Fc chimera protein (RnDsystems, 748-EC-050) was reconstituted at 100 μg/ml in sterile DPBS with Ca2+ and Mg2+ (Gibco, 14040-091). Protein A-coated PMMA microbeads (Microparticles, PMMA-Protein A-S2976B) 36 μm in diameter, were washed in 0.01% DPBS-T and incubated in 0.8 μg/ml Cdh1 solution at 1600 beads/ml for 90 min at 4°C with 1400 rpm mixing (Thermomixer, Eppendorf). After washing with 0.01% DPBS-T, microbeads were incubated in 1% heat-inactivated BSA (80°C, 10 min; Sigma, A3311) overnight at 4°C to block non-coated sites.
7
Analyzing BrdU Incorporation in Morulae and Blastocysts
8
Murine Oocyte Microinjection and Imaging
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Wild-type mice were purchased from CLEA Japan or Japan SLC. Female B6D2F1 mice (>8 wk old) were hyperovulated by injecting 5 U of human chorionic gonadotropin (hCG) 48 h after injecting 0.1 ml CARD HyperOva (Kyudo Company). Eggs were collected from the oviducts and placed in TYH medium (119.37 mM NaCl, 4.78 mM KCl, 1.19 mM KH2PO4, 5.56 mM glucose, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, 1.71 mM CaCl2, 1.19 mM MgSO4, 25.07 mM NaHCO3, 4 mg/ml BSA [ALBMAX I, Gibco; or A3311, Sigma-Aldrich]), potassium-supplemented simplex optimized medium (KSOM), or M2 medium at 37°C and 5% CO2. To remove cumulus cells, eggs were treated with 1 mg/ml hyaluronidase (Sigma-Aldrich) for 5 min at 37°C. For Juno/CD9 immunofluorescence and EM experiments, the zona pellucida was removed by treatment with 1 mg/ml collagenase (Sigma-Aldrich) for 5 min. In vitro mRNA transcription was performed using a mMessage mMachine T7 Kit (Ambion). After microinjection of mCherry-MBD-NLS (1.2 pg) or 3mEGFP_UtrCH (1.2 pg) mRNA using a micromanipulator (Narishige) and a piezo-driven pipette (Prime Tech) on an inverted microscope (Olympus), eggs were cultured for 2 h before imaging. His-Ran was diluted to a final concentration of 1.5 µg/µl (in PBS containing 1 mM DTT and 0.05% Tween 20), and 50 pl was used for injection.
9
Murine Embryo Production Protocol
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Six- to twelve-week-old ICR female mice (Japan SLC, Shizuoka, Japan) were superovulated by injection of 7.5 IU of equine chorionic gonadotropin (eCG; ASUKA
Animal Health, Tokyo, Japan) followed 46–48 h later by 7.5 IU of human chorionic gonadotropin (hCG; ASUKA Animal Health). Cumulus-oocyte complexes (COCs) were
collected from the ampullae of the excised oviducts 14 h after the hCG injection. COCs were placed in a 100-μl droplet of human tubal fluid (HTF) medium
supplemented with 4 mg/ml BSA (A3311; Sigma-Aldrich, St. Louis, MO, USA) [26 (link)]. Spermatozoa were collected from the cauda
epididymis of 12- to 18-week-old ICR male mice (Japan SLC) and cultured for at least 1 h in a 100-μl droplet of HTF. After preincubation, sperm suspension was
added into fertilization droplets at a final concentration of 1 × 106 cells/ml. At 2 hours post-insemination (hpi), morphologically normal fertilized
embryos were collected and cultured in potassium simplex optimized medium (KSOM) supplemented with amino acids [27 (link)] and 1
mg/ml BSA under paraffin oil (Nacalai Tesque, Kyoto, Japan). All incubations were performed at 37°C under 5% CO2 in air.
Animal Health, Tokyo, Japan) followed 46–48 h later by 7.5 IU of human chorionic gonadotropin (hCG; ASUKA Animal Health). Cumulus-oocyte complexes (COCs) were
collected from the ampullae of the excised oviducts 14 h after the hCG injection. COCs were placed in a 100-μl droplet of human tubal fluid (HTF) medium
supplemented with 4 mg/ml BSA (A3311; Sigma-Aldrich, St. Louis, MO, USA) [26 (link)]. Spermatozoa were collected from the cauda
epididymis of 12- to 18-week-old ICR male mice (Japan SLC) and cultured for at least 1 h in a 100-μl droplet of HTF. After preincubation, sperm suspension was
added into fertilization droplets at a final concentration of 1 × 106 cells/ml. At 2 hours post-insemination (hpi), morphologically normal fertilized
embryos were collected and cultured in potassium simplex optimized medium (KSOM) supplemented with amino acids [27 (link)] and 1
mg/ml BSA under paraffin oil (Nacalai Tesque, Kyoto, Japan). All incubations were performed at 37°C under 5% CO2 in air.
10
Deionized Protein Stock Preparation
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Stock solutions of d-BSA, deionized HSA (d-HSA) and deionized BGG (d-BGG) were prepared as described
previously [6 , 8 (link)]. BSA (A3311,
Sigma-Aldrich, St. Louis, MO, USA), HSA (A1653, Sigma-Aldrich) or BGG (G5009, Sigma-Aldrich) was dissolved in
CZB medium [9 (link)] deprived of BSA and EDTA at a concentration of 12%.
Approximately 3 g of mixed ion-exchange resin beads (501-X8(D); Bio-Rad Laboratories, Hercules, CA, USA) was
then added to 10 ml of each solution, and the mixture was incubated at room temperature with occasional
stirring. When they changed color from blue-green to gold, the BSA, HSA and BGG solutions were transferred to
fresh beads for a total of three exchanges. The supernatants were collected and stored at 4 C as stock
solutions.
previously [6 , 8 (link)]. BSA (A3311,
Sigma-Aldrich, St. Louis, MO, USA), HSA (A1653, Sigma-Aldrich) or BGG (G5009, Sigma-Aldrich) was dissolved in
CZB medium [9 (link)] deprived of BSA and EDTA at a concentration of 12%.
Approximately 3 g of mixed ion-exchange resin beads (501-X8(D); Bio-Rad Laboratories, Hercules, CA, USA) was
then added to 10 ml of each solution, and the mixture was incubated at room temperature with occasional
stirring. When they changed color from blue-green to gold, the BSA, HSA and BGG solutions were transferred to
fresh beads for a total of three exchanges. The supernatants were collected and stored at 4 C as stock
solutions.
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