We used 4% paraformaldehyde/PBS at room temperature for 45 minutes to fix cells and permeabilization of cells was done by 0.2% Triton X-100 in PBS. The cells were blocked overnight with a blocking solution (5% milk and 0.05% Tween-20 in PBS) and then incubated with primary antibodies by using 1:100 dilution of monoclonal anti-DDX4 (ab13840, Abcam, USA), monoclonal anti-DAZL (ab34139, Abcam, USA), as a germ cell markers and monoclonal anti-SCP3 (ab15093, Abcam, Cambridge, UK), monoclonal anti-GDF9(ab93892, Abcam, USA) and monoclonal anti-GDF9B (ab108413, Abcam, Cambridge, UK) as oocyte like cells markers for 2.5 hrs. Afterward, cells were washed in PBS/Tween 20 (0.1%) (Sigma) three times and incubated with Alexa fluor 488 and 594 secondary antibodies (Sigma, USA) for 1hrs in a dark place at room temperature. Cells were again washed with PBS/Tween 20 (0.1%) three times. Finally, the cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, D8417) and studied by Olympus DP73 digital camera associated with a fluorescence microscopy IX81 (U-MW-IB3). ImageJ software was used to assess the proportion of DDX4, DAZL, SCP3, GDF9, and GDF9B proteins in the resultant microphotographs.
Ab93892
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab93892 is a primary antibody used for detection of the target protein. It is suitable for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab13840, by Abcam (2 mentions)
Pa5 34401, by Thermo Fisher Scientific (2 mentions)
Ab15093, by Abcam (1 mentions)
Ab34139, by Abcam (1 mentions)
Dp73 digital camera, by Olympus (1 mentions)
U mwib3, by Olympus (1 mentions)
Ab134154, by Abcam (1 mentions)
Ab150080, by Abcam (1 mentions)
Ab150081, by Abcam (1 mentions)
D3571, by Thermo Fisher Scientific (1 mentions)
P9416, by Merck Group (1 mentions)
T8787, by Merck Group (1 mentions)
Af7021, by Affinity Biosciences (1 mentions)
Iseq00010, by Merck Group (1 mentions)
Ripa lysis solution, by Beyotime (1 mentions)
Beyoecl plus kit, by Beyotime (1 mentions)
Goat anti rabbit fluorescein isothiocyanate conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Te2000 s, by Nikon (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Ab6717, by Abcam (1 mentions)
5 protocols using ab93892
1
Immunofluorescence Characterization of Germ Cell and Oocyte Markers
2
Immunostaining of Oocyte Proteins
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In general, at room temperature, oocytes were fixed with 4% paraformaldehyde in PBS for 30min, and permeabilized with 0.3% Triton-100 (T8787, Sigma-Aldrich) in PBS for 20min, then blocked with 1% BSA (0332, amresco) in PBS containing 0.1% Tween-20 (P9416, Sigma-Aldrich) and 0.01% Triton − 100 for 30min. Next, oocytes were incubated with primary antibodies in 1% BSA for 1h. After being washed three times in PBS containing 0.1% Tween-20 and 0.01% Triton − 100 with each time for 5min, oocytes were incubated with secondary antibody in PBS containing 0.1% Tween-20 and 0.01% Triton − 100 for 30min. Similarly, after being washed three times, oocytes were transferred onto slides in PBS containing 0.1% Tween-20 and 0.01% Triton − 100 and detected under a laser scanning confocal microscope (Dragonfly, Andor Technology, UK).
The primary antibodies were listed as follows: MTX2 (1:100 in dilution, 11610-1-AP, Proteintech), GDF9 (1:100 in dilution, ab93892, Abcam), TACC3 (1:50 in dilution, ab134154, Abcam), EIF5A (1:50 in dilution, 11309-1-AP, Proteintech), ASPM (1:150 in dilution, 26223-1-AP, Proteintech). The secondary antibodies were presented as follows: Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) preadsorbed (1:500 in dilution, ab150081, Abcam), Goat Anti-Rabbit IgG H&L (Alexa Fluor 594) (1:500 in dilution, ab150080, Abcam), DAPI (1:500 dilution, D3571, Life Technologies).
The primary antibodies were listed as follows: MTX2 (1:100 in dilution, 11610-1-AP, Proteintech), GDF9 (1:100 in dilution, ab93892, Abcam), TACC3 (1:50 in dilution, ab134154, Abcam), EIF5A (1:50 in dilution, 11309-1-AP, Proteintech), ASPM (1:150 in dilution, 26223-1-AP, Proteintech). The secondary antibodies were presented as follows: Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) preadsorbed (1:500 in dilution, ab150081, Abcam), Goat Anti-Rabbit IgG H&L (Alexa Fluor 594) (1:500 in dilution, ab150080, Abcam), DAPI (1:500 dilution, D3571, Life Technologies).
3
Protein Expression Analysis in Ovarian Tissues
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Reference was made to previously developed protocols 55 (link), 75 (link); proteins from six ovaries were extracted with RIPA lysis solution (Beyotime, P0013C). After denaturation treatment, the protein of each sample was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF, Millipore, ISEQ00010, USA) membrane. Subsequently, protein bands were blocked at a temperature of approximately 20 ℃ for 2 h, and the primary antibody was incubated at 4 ℃ overnight. The following day, after three washes, the secondary antibodies were incubated. Finally, a BeyoECL Plus kit (Beyotime, P0018) was used for chemiluminescence. The primary antibodies used for Western blot consisted of MVH (Abcam, ab13840), GDF9 (Abcam, ab93892), LHX8 (Abcam, ab137036), BMPR1A (Affinity, DF6634, Shanghai, China), SMAD3 (Abcam, ab40854), and GAPDH (Affinity, AF7021).
4
Immunofluorescence Staining of Porcine Oocytes
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Immunofluorescence staining was performed as previously described by Lee et al. [22 (link)]. Porcine oocytes were washed three times in PBS containing 0.1% PVA and fixed with 4% paraformaldehyde (PFA). After washing three times in PBS, the oocytes were placed in 1% Triton X-100 for 30 min and incubated in 2% BSA-PBS to block non-specific sites overnight at 4 °C. Porcine oocytes were incubated with rabbit polyclonal antibodies against GDF9 (ab93892; Abcam, Cambridge, UK) and BMP15 (PA5-34401; Invitrogen) at 37 °C for 2 h. The samples were washed three times in 2% BSA and then incubated with a goat anti-rabbit fluorescein isothiocyanate-conjugated secondary antibody (1:200; Jackson Immuno Research Laboratories Inc., West Grove, PA, USA) for 2 h. Oocytes were mounted on glass slides and the images were captured using an epifluorescence microscope (TE2000-S; Nikon). Fluorescence intensities were measured using ImageJ software (version 1.46 r; National Institutes of Health).
5
Immunofluorescence Analysis of Oocyte Markers
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Immunofluorescence staining was performed to evaluate the effect of ASC and ASC-CM on the expression of GDF9 and BMP15 in oocytes. In brief, oocytes were washed three times in PBS containing 0.2% PVA. Then, they were fixed with 4% paraformaldehyde (w/v) in PBS for 30 min at room temperature. After incubation, the fixed oocytes were washed three times in PBS, and oocytes were permeated with 1% (v/v) Triton X-100 in PBS for 2 h at room temperature. The samples were washed and blocked with 2% bovine serum albumin in PBS for 4 h at 4 °C. The primary antibodies for GDF9 (1:200; ab93892, Abcam, MA, USA) and BMP15 (1:200; PA5–34401, Thermo Fisher Scientific, IL, USA) were treated to the oocytes, and incubation was performed at 37 °C overnight. After washing three times in PBS with 2% BSA, they were incubated with a secondary antirabbit polyclonal antibody (1:200; ab6717, Abcam) for 3 h at room temperature. The samples were washed several times with PBS and they were mounted on glass slides. Images were captured under a fluorescence microscope (Nikon Corp.) with the same exposure times and adjustments at magnification ×100. The fluorescence intensities of GDF9 and BMP15 were measured by Image J software (version 1.46r; National Institutes of Health, USA).
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