Normal villi tissues and CHM tissues were voluntarily donated by women who underwent surgical abortion. After gender identification, the trophoblastic tissue sections were immunostained with the primary antibodies (H3K27me3, Abcam, ab192985, 1:1000; USP21, Abcam, ab38864, 1:200; Nanog, Abcam, ab109250, 1:1000), as discussed before 17 (link) and examined using confocal microscopy (Olympus, Tokyo, Japan).
Ab38864
Manufactured by Abcam
Sourced in United Kingdom
Ab38864 is a lab equipment product. It is a multipurpose device designed for various laboratory applications. The core function of this product is to perform specific tasks required in a research or analytical setting.
Automatically generated - may contain errors
Lab products found in correlation
Ab109250, by Abcam (2 mentions)
Ab192985, by Abcam (2 mentions)
Confocal microscopy, by Olympus (1 mentions)
Ab207298, by Abcam (1 mentions)
Ab21624, by Abcam (1 mentions)
Ab97959, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Ecl chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Ab19857, by Abcam (1 mentions)
Radioimmunoprecipitation assay (ripa), by Thermo Fisher Scientific (1 mentions)
Ab9485, by Abcam (1 mentions)
Sc 28310, by Santa Cruz Biotechnology (1 mentions)
K5007, by Agilent Technologies (1 mentions)
Image quant las400 mini, by GE Healthcare (1 mentions)
Ab8245, by Abcam (1 mentions)
Bradford protein assay kit, by Beyotime (1 mentions)
Benchmark xt, by Roche (1 mentions)
Spss 18.0 statistical software, by IBM (1 mentions)
5 protocols using ab38864
1
Immunostaining of Trophoblastic Tissues
2
Profiling Stemness and Proliferation Markers
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Cells or tissue samples were washed by PBS for four times. Then, the protein was separated by RIPA (Thermo Scientific), electrophoresed to SDS-PAGE, and transferred onto PVDF membranes. After, protein was identified by specific primary antibodies USP21 (ab38864, 1 : 1000; Abcam), Oct4 (ab19857, 1 : 2500; Abcam), Nanog (ab21624, 1 : 3000; Abcam), Sox2 (ab97959, 1 : 2500; Abcam), FOXM1 (ab207298, 1 : 1500; Abcam), and GAPDH (ab9485, 1 : 3000; Abcam) overnight at 4°C. Then, HRP-conjugated goat anti-rabbit IgG secondary antibody (ab205718, 1 : 2000; Abcam) was applied to the membranes, and the bands were visualized by ECL chemiluminescence reagent (Thermo Scientific). The gray level of each band was quantitatively analyzed by ImageJ software.
3
Immunohistochemical Analysis of USP21 and Fra-1
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Paraffin-embedded tissue blocks were sectioned at 4 μm thickness, dewaxed in xylene, and then rehydrated in a graded alcohol series. Endogenous peroxidase was blocked with 3% hydrogen peroxide. Tissue sections were immersed in 10 mM citrate buffer (pH 6.0), rinsed in Tris-buffered saline, and then heated in a microwave oven for three 5-min cycles. Sections were stained with hematoxylin and eosin (H&E) to examine tumor morphology before applying the primary antibody. Then, USP21 and Fra-1 were detected with the following primary antibodies: anti-USP21 (ab38864, Abcam, Cambridge, UK) and anti-Fra-1 (SC-28310, Santa Cruz Biotechnology), and sections were subsequently treated with secondary antibody for 30 min and incubated with avidin-biotin-peroxidase complex for 30 min (K5007, Dako, Carpinteria, CA, USA). Diaminobenzidine was used as the chromogen along with slight hematoxylin counterstaining.
4
Western Blot Analysis of Trophoblastic Proteins
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Trophoblastic tissues were lysed in RIPA buffer. The protein concentration was measured using the Bradford Protein Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. Western blot was performed as previously described 16 (link). The membranes were incubated with primary antibodies (all from Abcam, Cambtidge, UK) against H3K27me3(ab192985, 1:1000), USP21(ab38864, 1:200), Nanog (Abcam, ab109250, 1:1000), and GAPDH (Abcam, ab8245, 1:10000). The bands were detected with FDbio-Dura ECL (FUDE, Zhejiang, China) using the Imagequant LAS400mini (GE Healthcare, Buckinghamshire, UK). Image J software (NIH, Bethesda, MD) was used to measure the gray value of each band. The signal of each band was normalized to that of GAPDH in the same membrane before comparison.
5
Immunohistochemical Evaluation of USP21
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Sections of the TMA were immunohistochemically labeled with USP21 (Abcam, ab38864, 1:100) using a BenchMark XT automated stainer (Ventana Medical Systems, Inc., Oro Valley, AZ, USA), according to the manufacturer’s protocol. To evaluate expression of USP21, an IHC score was generated by multiplying the percentage of USP21-positive cells by staining intensity. Staining intensity categories were as follows: negative, score of 0; mildly positive, score of 1; moderately positive, score of 2; and strongly positive, score of 3. For example, when all cancer cells were stained with maximal intensity (3+), then the IHC score was 300 (100% × 3). Statistical analyses were performed using SPSS 18.0 statistical software (SPSS). Chi-squared tests (Pearson’s Chi-squared test or Chi-squared test using linear by linear association) were used to analyze correlations between IHC results and clinicopathologic parameters. p-Values below 0.05 were considered statistically significant. Survival curves were plotted using the Kaplan–Meier method.
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