Oocytes were fixed in 4% formaldehyde solution (P0099, Beyotime, China) at room temperature for 30 min. After being washed 3 times using 0.1% polyvinyl alcohol (PVA)-PBS, the oocytes were permeabilized in 0.5% Triton X-100 diluted with Dulbecco’s Phosphate Buffered Saline (D-PBS) at room temperature for 20 min. For immunostaining, samples were first blocked with 1% bovine serum albumin (BSA, A1933, Sigma-Aldrich, United States) at room temperature for 1h, and then incubated at 4°C for 2 h with a FITC-conjugated anti-α-tubulin mouse monoclonal antibody (1:500, F2168, Sigma-Aldrich, United States), which specifically recognizes α-tubulin (Zhu et al., 2018 (link); Han et al., 2020 (link)). After being washed three times with D-PBS at room temperature in the dark, nuclei were stained with DAPI (10ug/ml, E607303, Sangon, China) at room temperature for 10 min. Oocytes were mounted on glass slides with antifade solution (S2100, Solarbio, China). For each oocyte to be imaged, the top and the bottom were determined and 7 evenly spaced z-sections were captured using z-stacks under a rotary laser confocal microscope (Dragonfly, Andor Technology) driven by Fusion Software. Images were presented as maximum intensity projections of the middle 5 frames (excludes the first one and the last one since these two sections have little information).
F2168
Manufactured by Merck Group
Sourced in United States
The F2168 is a laboratory equipment product manufactured by the Merck Group. It is a versatile and reliable instrument designed for use in various scientific and research applications. The core function of the F2168 is to provide accurate and consistent measurements for specific parameters required in laboratory settings. However, a detailed description of the intended use or specific applications of this product cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (4 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Lsm 700, by Zeiss (2 mentions)
P0099, by Beyotime (1 mentions)
E607303, by Sangon (1 mentions)
S2100, by Solarbio (1 mentions)
Ab179503, by Abcam (1 mentions)
Ab187139, by Abcam (1 mentions)
Ab28193, by Abcam (1 mentions)
Lsm 900 meta, by Zeiss (1 mentions)
T7451, by Merck Group (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
A11058, by Thermo Fisher Scientific (1 mentions)
A11055, by Thermo Fisher Scientific (1 mentions)
Geminin, by Santa Cruz Biotechnology (1 mentions)
Ab11316, by Abcam (1 mentions)
Ab113692, by Abcam (1 mentions)
Be2011, by EASYBIO (1 mentions)
Ac008, by ABclonal (1 mentions)
Zb 2305, by ZSGB-BIO (1 mentions)
16 protocols using f2168
1
Immunofluorescence Staining of Oocytes
2
Spindle and Chromosome Analysis of MII Oocytes
3
Immunofluorescence Analysis of Oocyte Proteins
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Denuded oocytes were fixed in 4% paraformaldehyde/PBS (fixation solution) for 30 minutes and then transferred to 1% Triton X‐100 (permeabilization solution) for 8 hours. After washing in PBST, oocytes were incubated with 3% BSA/PBS (blocking solution) for 1 hour at room temperature (RT), followed by incubation with rabbit anti‐HDAC8 antibody (1:50; ab187139; Abcam), mouse anti‐α‐tubulin‐FITC antibody (1:200; F2168; Sigma‐Aldrich), rabbit anti‐γ‐tubulin antibody (1:50; ab179503; Abcam), sheep anti‐BubR1 antibody (1:50; ab28193; Abcam) or mouse anti‐acetylated‐α‐tubulin (Lys 40) antibody (1:50; T7451; Sigma‐Aldrich) at 4°C overnight. After three times of washes in PBST, oocytes were incubated with corresponding secondary antibodies for 1 hour and counterstained with 10 µg/mL propidium iodide (PI) or Hoechst 33342 for 10 minutes at RT. Finally, oocytes were mounted on the glass slides and imaged by the laser confocal microscope (LSM 900 META, Zeiss).
4
Immunofluorescence Staining of Oocytes and Embryos
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Oocytes and embryos were fixed for 1 h in 3.7% paraformaldehyde in PBS and permeabilized with 0.5% Triton X-100 in PBS for 20 min at room temperature. Then the cells were incubated at 4°C overnight with primary antibodies (geminin at 1:50 [sc8449; Santa Cruz Biotechnology, Dallas, TX], Pi-H2AXS139 [9718] and Pi-Chk1S345 [2348] at 1:200 [Cell Signaling Technology], and Pi-Chk2T68 at 1:200 [BS4043; Bioworld]) and then incubated for 1 h with a secondary Alexa Fluor 488–conjugated antibody (1:1000; A11008 and A11055; Life Technologies) or Alexa Fluor 594–conjugated antibody (1:1000; A11058; Life Technologies). For α-tubulin staining, the oocytes were incubated with only the anti–α-tubulin–fluorescein isothiocyanate antibody (1:1000; F2168; Sigma-Aldrich) for 2 h at room temperature. DNA was stained for 20 min with 4′,6-diamidino-2-phenylindole (DAPI) or propidium iodide (PI). Fluorescence was detected using a Zeiss LSM710 laser-scanning confocal microscope.
5
Immunofluorescence Imaging of Oocyte Microtubules
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Oocytes were collected and fixed with 4% (w/v) paraformaldehyde in PBS (pH 7.4) for 40 min at room temperature. Fixed samples were permeabilized in incubation buffer (0.5% Triton X-100 in 20 mM Hepes, pH 7.4, 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose) for 30 min. After washing twice in PBS containing 0.01% Triton-X100, samples were incubated in block solution (PBS containing 1% BSA) for 1 h at RT. The microtubules were localized by incubation for 1 h with a fluorescein isothiocyanate-labeled mouse monoclonal antibody against α-tubulin (Sigma Chemical Co., F-2168), which was diluted 1∶100 in blocking solution. Nuclear statuses of oocytes were evaluated by staining with 10 mg/mL propidium iodide (PI) in PBS for 10 min. Finally, the samples were observed under a Zeiss confocal laser scanning microscope. The area of the spindle was counted using Image ProPlus software.
6
Antibody Staining Protocol for Cell Imaging
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Mouse antibody to α‐Tubulin‐FITC (1:400, F2168) was purchased from Sigma (USA). Mouse antibodies to γ‐Tubulin (1:200, ab11316) and TPM3 (1:100, ab113692) were purchased from Abcam (USA). Mouse antibody to FLAG (1:2000, M20008L) was purchased from Abmart (Shanghai, China). Rabbit antibody to MYC (1:1000, BE2011) was purchased from EASYBIO (Beijing, China). Rabbit antibodies to β‐Actin (1:2000, AC026) and β‐Tubulin (1:1000, AC008) were purchased from ABclonal (Wuhan, China). Rabbit antibodies to RNF20 (1: 1000, 21625‐1‐AP) and TPM3 (1:1000, 10737‐1‐AP) were purchased from Proteintech Group (USA). Rabbit antibodies to H2Bub (1: 1000, 5546s), H2B (1: 1000, 12364S) and H3 (1: 1000, 4499S) were purchased from Cell Signaling Technology (USA). Mouse antibody to HEC1 (1: 100, sc‐515510) was purchased from Santa Cruz Biotechnology (USA). Rabbit antibodies to RBBP7 (1:100, bs8620) and UBASH3B (1:100, bs8741) were purchased from Bioworld (Beijing, China). Human antibody to ACA (1: 200, 15–234) was purchased from Antibodiesinc (USA). Goat anti‐rabbit FITC (1:200, ZF‐0311) and goat anti‐mouse TRITC (1:200, ZB‐2305) were purchased from ZSGB‐BIO (Beijing, China). Alexa Fluor 680‐conjugated goat anti‐mouse (1:10000, A21057) and Alexa Fluor 800‐conjugated goat anti‐rabbit (1:10000, A21109) were purchased from Invitrogen (USA).
7
Immunofluorescence Staining of Oocytes
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Human or mouse oocytes were fixed in 3.7% paraformaldehyde diluted in PBS for 30 min at room temperature and then permeabilized with 0.2% TritonX‐100 for 15 min. After incubation for 1 h in blocking buffer (1% BSA diluted in PBS with 0.1% TritonX‐100), oocytes were stained with the indicated primary antibodies diluted in blocking buffer overnight at 4°C. After three washes, samples were incubated with Alexa Fluor 568‐conjugated goat anti‐rabbit (A11036, Invitrogen), Alexa Fluor 488‐conjugated goat anti‐rabbit (A11001, Invitrogen), Alexa Fluor 568‐conjugated donkey anti‐mouse (A10037, Invitrogen) secondary antibodies with dilution of 1:300, in combination with 1 µg/ml DAPI (236276, Roche, Basel, Switzerland), with or without FITC‐α‐tubulin (1:400, F2168, Sigma‐Aldrich), or Alexa Fluor 647‐conjugated phalloidin (1:200, A22287, Invitrogen) for 1 h at room temperature. After washing four times, the oocytes were mounted on slides with anti‐fade medium and imaged using a laser‐scanning confocal microscope (LSM800, Carl Zeiss, Jena, Germany). The primary antibodies used were as follows: mouse monoclonal anti‐FLAG (1:500, F1804, Sigma‐Aldrich), rabbit monoclonal anti‐pERK1/2 (1:400, #4370, Cell Signaling Technology, Danvers, MA, USA), and rabbit polyclonal anti‐TPX2 (1:200, NB500‐179, Novus Biologicals, Littleton, CO, USA).
8
Dynamics of Cytoskeletal Structures during Polar Body Extrusion
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To observe the microtubule, microfilament and chromosome dynamics during p2PB extrusion, piPSNT embryos were sampled from control (no 6DMAP) treatment and 6DMAP treatment groups at 0, 1.5, 4 and 8 hpa, respectively. They were fixed with 4% paraformaldehyde for 40 min, then permeabilized with 1% Triton X-100 for 30 min. After incubation in Image-iTTM FX Signal Enhancer (I36933, Invitrogen, Carlsbad, CA) for 30 min, the embryos were further blocked with 1% bovine serum albumin (A9418, Sigma, St. Louis, MO) in PBS for 1 h. They were then incubated with TRITC conjugated phalloidin (1:200, P1951, Sigma, St. Louis, MO) at 37°C for 2 h. After completely washing and secondary blocking, they were further incubated in FITC conjugated monoclonal anti-α-tubulin (1:200, F2168, Sigma, St. Louis, MO) for another 3 h. After the nuclei were stained with 10 μg/ml Hoechst 33342 (B2261, Sigma, St. Louis, MO), the embryos were mounted with anti-fade reagent (S36937, Invitrogen, Carlsbad, CA) and observed under laser-scanning confocal microscope (Zeiss, LSM700).
9
Immunostaining of Oocyte DNA Damage
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Oocytes were washed with phosphate-buffered saline (PBS) (P4417, Sigma-Aldrich), fixed with 3.7% paraformaldehyde (w/v) in PBS containing 0.1% PVA (PBS-PVA), and permeabilized with 1% Triton X-100 (v/v) for 30 min at 37°C. After incubation in blocking buffer (PBS containing 1% BSA) for 1 h, the samples were incubated overnight with different antibodies in a blocking buffer at 4°C and washed three times with PBS containing 1% BSA. The antibodies used were rabbit anti-RAD51 (sc-8349, 1:100; Santa Cruz), mouse anti-γH2AX (ab26350, 1:100; Abcam), rabbit anti-ATM (pS1981, 1:100; Cell Signaling Technology), goat anti-CHK1 (pS345, 1:100; Santa Cruz), anti-α-tubulin conjugated to FITC (F2168, 1:100; Sigma-Aldrich), and anti-BUB3 (mitotic checkpoint protein BUB3; sc-28258, 1:100; Santa Cruz). Stained RAD51 oocytes were treated with 0.1% pronase for 30 sec to expand the zona pellucida and then centrifuged for 10 min for removal of the lipid droplets prior to fixation. The oocytes were washed three times with PBS-PVA, and then labeled with a FITC-conjugated antibody (1:100) for 1 h at room temperature. The oocytes were counterstained with 5 μg/ml Hoechst 33342 (Sigma Life Science) for 15 min, mounted on a glass slide, and examined using an LSM 710 META confocal laser-scanning microscope (Zeiss, Jena, Germany).
10
Immunofluorescence analysis of meiotic spindle
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Oocytes (3 and 6 h after GVBD) were transferred in a precooling M2 solution (4°C) for 4 min. Then, oocytes were fixed in a PBS solution containing 5% formaldehyde and 0.3% Triton X-100 at room temperature for 15 min. Oocytes were blocked in 950 μl PBS containing 30 mg BSA and 50 μl 1% Tween20 for 1 h. Then, oocytes were incubated in primary antibody for 1 h at 37°C: human anti-centromere (Immunovision, HCT-0100, 1:200), mouse monoclonal anti-alpha-tubulin with FITC (Sigma, F2168, 1:400), rabbit polyclonal anti-pS55-Hec1 (Genetex, GTX70017, 1:200), and rabbit polyclonal anti-Mad2 (Biolegend, PRB-452C, 1:400). After rinsing three times in PBS, oocytes were transferred into secondary antibodies: anti-human-Cy3 (Jackson ImmunoResearch, AB-2340538, 1:200) and Goat Anti-Rabbit IgG with Fluor 488-labeled (Beyotime, A0423, 1: 400) for 1.5 h at 37°C. Hoechst 33342 (Invitrogen, H21492, 50 mg/mL) was used for Chromosomes stain. Images were acquired using a Nikon A1 confocal laser-scanning microscope (Nikon, Tokyo, Japan) with a 100× objective, scanning z-axis at 0.4 mm intervals to span the entire region of the spindle.
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