Paired ovaries were collected at 6 h after ovarian resection to detect the expression of p-rpS6, Foxo3a and NGF. Briefly, 5 μm sections were deparaffinized, rehydrated and endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide in methanol for 20 min. Antigen retrieval pretreatment was carried out by boiling the sections in 0.01 M citrate buffer, pH 6.0 for 10 min. Immunohistochemical analyses were performed using a Histostain Kit (856743, Invitrogen, Carlsbad, CA, USA) with antibodies against p-rpS6 (S235/236) (4858, Cell Signaling Technology, Beverly, MA, USA), Foxo3a (ab53287, Abcam, Cambridge, MA, USA) and NGF (ab6199, Abcam) overnight at 4 °C. For some sections, primary antibodies were replaced with non-immune rabbit IgG as negative controls.
Ab53287
Manufactured by Abcam
Sourced in United States
Ab53287 is a laboratory equipment product offered by Abcam. It is a device designed for specific experimental purposes. No further details about its core function or intended use can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab154786, by Abcam (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Ab6199, by Abcam (1 mentions)
Histostain kit, by Thermo Fisher Scientific (1 mentions)
S235 236, by Cell Signaling Technology (1 mentions)
Bicinchoninic acid assay kit, by Merck Group (1 mentions)
Ubiquitin, by Abcam (1 mentions)
Ab111943, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab7780, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bcl 2, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Ab32124, by Abcam (1 mentions)
Foxo3a, by Abcam (1 mentions)
Sc 7985, by Santa Cruz Biotechnology (1 mentions)
A3854, by Merck Group (1 mentions)
Sc 8312, by Santa Cruz Biotechnology (1 mentions)
Sc 74513, by Santa Cruz Biotechnology (1 mentions)
Anti mouse igg peroxidase a9044, by Merck Group (1 mentions)
7 protocols using ab53287
1
Ovarian Protein Expression Analysis
2
Protein Expression Analysis of TRIM69 and Apoptosis Markers
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The specimens and HLECs were lysed with RIPA lysis buffer at 4 °C. A bicinchoninic acid assay kit (Sigma Chemical Co., St. Louis, MO) was used for protein quantification. Proteins of each sample (25 μg per sample) were subjected to 10% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot assays were performed with primary antibodies to the following: TRIM69 (Ab111943; Abcam, Cambridge, MA, USA); p53 (Ab26; Abcam); Bax (Ab32503; Abcam); Bcl-2 (Ab32124; Abcam); Foxo3a (Ab53287; Abcam); ubiquitin (Ab7780; Abcam); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#5174; Cell Signaling Technology, Danvers, MA, USA). GAPDH protein was used as the loading control.
3
Endothelial Cell Culture and Signaling
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Endothelial Cell Growth Medium (211-500) was purchased from the European Collection of Authenticated Cell Cultures (ECACC, Porton Down, UK). DMEM/F12 (Sigma 51445C), antibiotic mixture, fetal bovine serum, trypsin–EDTA were purchased from Sigma-Aldrich Ltd (Poznan, Poland). The antibodies against ferritin H (sc-25617) and L (sc-74513), p-Akt (Ser-473) (sc-7985-R), Akt (sc-8312), siRNA Akt 1, 2, 3 (sc-2925; sc-29197; sc-38912), control (sc-37007) and all the reagents for siRNA transfection were obtained from Santa Cruz Biotechnology (Dallas, USA). Antibodies against FOXO3a (ab53287) and p-FOXO3a (Ser 253) (ab154786) were from Abcam (Cambridge, UK). Homocysteine (H4628), insulin (I9278), deferoxamine (DFO) (D9533), trypan blue solution (T8154), secondary antibodies anti-Rabbit IgG—Peroxidase (A9169) and anti-Mouse IgG—Peroxidase (A9044) as well as antibodies against β-actin (A3854) were from Sigma-Aldrich Ltd (Poznan, Poland).
4
Multiparametric Flow Cytometry Analysis
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Cells were isolated using Accutase (BioLegend), aliquoted (3×105 cells per condition) and fixed for 15 min in 4% (v/v, in PBS) paraformaldehyde (Electron Microscopy Sciences) at room temperature. For intracellular staining, cells were permeabilized by adding ice-cold 100% methanol slowly to prechilled cells while gently vortexing (to a final concentration of 90% methanol) and incubated at 4°C for 30 min. Then, cells were washed in PBS, centrifuged at 1200 g, and resuspended in PBS with 3% BSA (staining buffer). For immunostaining, cells were incubated in staining buffer containing a primary Ab specific to PD-L1 (CST, 13684) or FOXO3 (Abcam, ab53287) (1:200 dilution) or control rabbit IgG for 1 hour at room temperature. After washing three times with PBS, cells were incubated in staining buffer containing a FITC-conjugated antirabbit (1:200 dilution) secondary Ab (Jackson ImmunoResearch) for 30 min at room temperature in the dark. Subsequently, cells were characterized using a FACScan flow cytometer (BD Biosciences) at the Institutional shared flow cytometry facility. Ten thousand events were collected in each run. The data were analyzed by FlowJo software.
5
Antibodies for Western Blot and IHC Analysis
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Anti‐HIF‐1α (WB 1:500; IHC 1:100; ab51608), anti‐METTL3 (WB 1:1,000; IHC 1:500; ab195352), anti‐FOXO3 (WB 1:1,000; ab53287), goat anti‐rabbit IgG (WB 1:2,000; ab6721), and goat anti‐rabbit IgG‐FITC (IF 1:200, ab6717) antibodies were purchased from Abcam, USA. Anti‐MAP1LC3‐B (WB 1:1,000; IHC 1:100; IF 1:100; A7198), anti‐PDGF‐B (WB 1:1,000; A1195), anti‐VEGF‐A (WB 1:1,000; IHC 1:100; A5708), anti‐Ki67 (IHC 1:100; A11390), anti‐GAPDH (WB 1:1,000; AC027), anti‐tubulin (WB 1:5,000; AC021), anti‐β‐actin (WB 1:50,000; AC026), anti‐YTHDF1 (IHC 1:1,000; A18126), and anti‐YTHDF2 (IHC 1:1,000; A15616) antibodies were purchased from Abclonal, China. Anti‐m6A (MeRIP 1:1,000; ABE572) antibody was purchased from Merck Millipore (Massachusetts, USA). Western blot analyses were performed by standard methods described previously (Niu et al, 2019 ).
6
Quantifying HIF1α, IL-8, and AMPK Signaling
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Total RNA from cells and tissues was extracted with TRIzol (10296010, Thermo Fisher Scientific), and reverse transcription was performed with a reverse transcription kit (R323-01, Vazyme). qPCR was performed with SYBR (TSE202, Vazyme) in an Applied Biosystems QuantStudio 5 Real-Time PCR system. Finally, the relative expression was calculated using GAPDH as a standard. The primers for qPCR are listed in Supplemental Table 1 . Western blot analysis was conducted as reported previously. Briefly, cells and tissue samples were lysed in cell lysis buffer and tissue protein extraction reagent (78510, Thermo Fisher Scientific), respectively. A total of 40 μg protein was processed for subsequent analysis. Antibodies against HIF1α (36169T, Cell Signaling Technology), IL-8 (ab106350, Abcam), p-AMPK (2535T, Cell Signaling Technology), AMPK (ab32047, Abcam), FOXO3A (ab53287, Abcam), UQCRC2 (ab203832, Abcam), and tubulin (AF0001, Beyotime) were used.
7
Investigation of PI3K/Akt Signaling in Follicular Cells
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PN was purchased from Cayman Chemical. MG132 (one specific proteasome inhibitor) and NAC (N-acetyl-cysteine, one specific superoxide scavenger) were obtained from Sigma Aldrich. SC3036 (one specific PI3K/Akt agonist) was purchased from Santa Cruz Biotechnology. LY294002 (one specific PI3K/Akt inhibitor) was obtained from Cell Signaling Technology. FSH (human pituitary) was purchased from Merck Millipore. FSHR pAbs (orb213952) were obtained from Biorbyt. β-actin pAbs (YT0099), Lamin B1 pAbs (YT5180), p-Akt (T308) pAbs (YP0590) and Akt pAbs (YT0173) were purchased from Immunoway. NaK-ATPase mAbs (ab76020), 3-Nitrotyrosine mAbs (ab52309), control rabbit IgG (ab172730), Flag-tag pAbs (ab122902), p-FoxO3a (S253) mAbs (ab154786), FoxO3a mAbs (ab53287), Alexa-flour 488-conjugated secondary Abs (ab150077) and Cycloheximide (CHX, one specific protein synthesis inhibitor, ab120093) were obtained from Abcam.
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