For western blot analysis, total proteins were extracted using the Whole Cell Protein Extraction Kit (Key GEN, China). A BCA Protein Quantitation Assay (Thermo, USA) was used to measure the protein concentration. We separated protein samples by using 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) gels. The separated protein samples were then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). After blocking with 5% nonfat dry milk in Tris-buffered saline (TBS)/0.1% Tween 20 for 1 h at room temperature, the membranes were incubated with AKT (1:1000, 10176-2-AP, Proteintech), PI3K (1:1000, 60225-1-Ig, Proteintech), p-PI3K (1:1000, 28731-1-AP, Proteintech), p-PI3K (1:1000, ab182651, Abcam), PGK1 (1:1000, 17811-1-AP, Proteintech), Ser82 VIMENTIN (1:1000, ab52943-10, Abcam), Ser72 VIMENTIN (1:1000, ab52944, Abcam), Ser38 VIMENTIN (1:1000, ab52942, Abcam), Ser83 VIMENTIN (1:1000, 3878 T, CST, USA) and anti-GAPDH (1:1000, 5,174, CST, USA). The next day, membranes were washed 3 times with TBST buffer for 15 min and incubated with horseradish peroxidase-conjugated secondary antibody. Finally, the western blot signals were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA).
Ab52942
Manufactured by Abcam
Sourced in United States
Ab52942 is a polyclonal antibody specific for the detection of human FGFR2. This antibody is designed for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab52944, by Abcam (2 mentions)
Ab7763, by Abcam (2 mentions)
Ab16667, by Abcam (2 mentions)
Ab1316, by Abcam (2 mentions)
Bca protein quantitation assay, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Ab182651, by Abcam (1 mentions)
Whole cell protein extraction kit, by Keygen Biotech (1 mentions)
Ab215715, by Abcam (1 mentions)
Ab10433, by Abcam (1 mentions)
Gb12002, by Wuhan Servicebio Technology (1 mentions)
Ab32575, by Abcam (1 mentions)
Ab197202, by Abcam (1 mentions)
Ab217673, by Abcam (1 mentions)
Sc 13156, by Santa Cruz Biotechnology (1 mentions)
Dab substrate kit, by ZSGB-BIO (1 mentions)
4 protocols using ab52942
1
Comprehensive Western Blot Analysis Protocol
2
Protein Expression Analysis by Western Blot
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The expression of cytokeratin, vimentin, VEGF, LIF, and integrin β3 was analyzed by Western blotting. Briefly, equal amounts of proteins extracted from different groups were separated by 10% SDS-PAGE electrophoresis and transferred to PVDF membranes. The membranes were incubated with anti-cytokeratin (1:1,000 Abcam, ab7763), anti-vimentin (1:1,000, Abcam, ab52942), anti-VEGF (1:1,000, Abcam, ab1316), anti-TGF-β1 (1:1,000, Abcam, ab215715), and anti-ki67 (1:1,000, Abcam, ab16667) antibodies overnight at 4°C after blocking in 5% BSA and then incubated with HRP-conjugated goat anti-mouse or anti-rabbit secondary antibody at room temperature for 1 h on the second day. β-actin was used as a loading control.
3
Protein Expression Analysis Methods
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Primary antibodies used for Western blot, immunohistochemistry and immunocytochemistry are listed as followed: Hif-1α (79233, Cell signaling, USA), α-SMA (ab32575, Abcam, USA), Bnip3 (ab10433, Abcam, USA; ab38621, Abcam, USA), LC3B (83506, cell signaling, USA; 12741, cell signaling, USA), vimentin (ServiceBio, GB12192, China), phosphor-vimentin Ser56 (ab217673, Abcam, USA), phosphor-vimentin Ser38 (ab52942, Abcam, USA), phosphor-vimentin Ser72 (ab52944, Abcam, USA), phosphor-vimentin Ser82 (ab52943, Abcam, USA), caspase 3 (ab197202, Abcam, USA), cytochrome C (sc-13156, Santa Cruz, USA), GAPDH (GB12002, Servicebio, China).
4
Histological Analysis of Endometrial Injury
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Hematoxylin–eosin staining (H&E) was used to analyze the tissue structure. The uteri were fixed in 4% paraformaldehyde (PFA) solution overnight and embedded in paraffin, cut into 5-μm sections, and then stained with H&E.
To examine the collagen deposition in the injured endometrium, Masson’s trichrome staining was carried out. The extent of fibrosis among different groups, defined as the areas occupied by collagen fiber relative in proportion to the entire endometrium, was quantified by the software Image Pro Plus 6.0.
For immunohistochemistry staining (IHC), the slides were dewaxed in xylene and rehydrated through graded ethanol concentrations. Antigen retrieval was performed in citrate solution, and endogenous peroxidase activity was blocked in 3% H202 for another 15 min. The sections were incubated with anti-cytokeratin (1:200, Abcam, ab7763), anti-vimentin (1:200, Abcam, ab52942), anti-VEGF (1:200, Abcam, ab1316), and anti-ki67 (1:200, Abcam, ab16667) antibodies overnight at 4°C, followed by goat anti-mouse IgG or goat anti-rabbit IgG secondary antibody for 60 min, and then stained with DAB substrate kit (ZSGB-BIO, China) and hematoxylin in accordance with the manufacturer’s protocol.
To examine the collagen deposition in the injured endometrium, Masson’s trichrome staining was carried out. The extent of fibrosis among different groups, defined as the areas occupied by collagen fiber relative in proportion to the entire endometrium, was quantified by the software Image Pro Plus 6.0.
For immunohistochemistry staining (IHC), the slides were dewaxed in xylene and rehydrated through graded ethanol concentrations. Antigen retrieval was performed in citrate solution, and endogenous peroxidase activity was blocked in 3% H202 for another 15 min. The sections were incubated with anti-cytokeratin (1:200, Abcam, ab7763), anti-vimentin (1:200, Abcam, ab52942), anti-VEGF (1:200, Abcam, ab1316), and anti-ki67 (1:200, Abcam, ab16667) antibodies overnight at 4°C, followed by goat anti-mouse IgG or goat anti-rabbit IgG secondary antibody for 60 min, and then stained with DAB substrate kit (ZSGB-BIO, China) and hematoxylin in accordance with the manufacturer’s protocol.
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