Staining for ER, PR, HER2, and SHC1 was performed using Dako EnVision FLEX mini Kit on a Dako Autostainer Omnis (Agilent, Santa Clara, CA). High-resolution digital images were captured at × 20 using a Pannoramic 250 Flash III slide scanner (3DHISTECH Ltd., Budapest, Hungary). ER, PR, HER2, and SHC1 were detected using EnVision FLEX Target Retrieval Solution Low pH (Citrate buffer, pH 6.1, Agilent) and incubation with anti-ER (1:200, ab3575, Abcam, Cambridge, MA), anti-PR (1: 2000, ab16661, Abcam), anti-HER2 (1:400, A0485, Agilent), and anti-SHC (1:50, 06-203, Sigma) for 30 min, followed by secondary antibody (Polymer) for 30 min. Counterstaining with hematoxylin, dehydration, clearing, and coverslipping was performed. The Dako HER2 antibody recognizes rat HER2 [28 (link)].
Ab3575
Manufactured by Abcam
Sourced in United Kingdom
Ab3575 is a monoclonal antibody that recognizes the PCNA (Proliferating Cell Nuclear Antigen) protein. PCNA is a key component of the DNA replication and repair machinery in eukaryotic cells. Ab3575 can be used to detect and quantify PCNA expression in various experimental models.
Automatically generated - may contain errors
Lab products found in correlation
Ab3576, by Abcam (6 mentions)
Ab205718, by Abcam (2 mentions)
Las4000 enhanced chemiluminescence system, by GE Healthcare (2 mentions)
Ab16661, by Abcam (1 mentions)
Envision flex mini kit, by Agilent Technologies (1 mentions)
Pannoramic 250 flash 3 slide scanner, by 3DHISTECH (1 mentions)
A0485, by Agilent Technologies (1 mentions)
Citrate buffer, by Agilent Technologies (1 mentions)
Ab170933, by Abcam (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Ab5690, by Abcam (1 mentions)
Anti actin c 2, by Santa Cruz Biotechnology (1 mentions)
Anti f4 80, by Bio-Rad (1 mentions)
Bicinchoninic acid protein assay kit, by Tiangen Biotech (1 mentions)
Anti actin, by Abcam (1 mentions)
Anti rabbit mouse igg antibody, by CWBIO (1 mentions)
Sc 539, by Santa Cruz Biotechnology (1 mentions)
Beyoecl plus, by Beyotime (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Anti gapdh, by Abcam (1 mentions)
11 protocols using ab3575
1
Immunohistochemical Staining of ER, PR, HER2, and SHC1
2
Profiling ERα and FOXA1 Binding in Breast Cancer
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We used an immunotethered strategy for profiling the binding of the ERα and FOXA1 TF in human MCF7 breast cancer cells. MCF7 cells were estrogen-withdrawn for 72 hours before being plated and then treated with either ethanol (vehicle control) or 10−10 M E2 for 1 hour before cell collection. The CUT&RUN method uses an antibody to a specific chromatin epitope to tether protein A–MNase at chromosomal binding sites within permeabilized cells. The nuclease is activated by the addition of calcium and cleaves DNA around binding sites (10 (link)). Cleaved DNA is isolated and subjected to paired-end Illumina sequencing to map the distribution of the chromatin epitope genome-wide. We used a primary antibody to human ERα (ab3575, Abcam, Cambridge, MA) and human FOXA1 (ab170933) and protein A–MNase fusion (pA-MNase, a gift from S. Henikoff, Fred Hutchinson Cancer Research Center, Seattle WA) (10 (link)). CUT&RUN profiling with 5 × 105 cells and library amplification with 13 cycles of PCR were performed as described (10 (link)). Libraries were sequenced for 10 million paired-end reads on the Illumina NovaSeq 6000 platform at the University of Colorado Denver Cancer Center Genomics Shared Resource. Paired-end reads were mapped to the GRch38 assembly of the human genome using Bowtie2 (58 (link)).
3
Immunohistochemical Analysis of Tumor Biopsies
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A subset (Additional file 1 , n = 25) of the tumour biopsies were preserved in 10% neutral buffered formalin (NBF) for 24 h, paraffin embedded, and sections cut at 4 µm. Sections were stained with the following antibodies, ERα (1:300 dilution, ab3575) and CD3 (1:150 dilution, ab5690) (Abcam, Cambridge, UK). For CD3 evaluation, areas of stroma were identified and as many 40X fields of view were examined as possible (range 3–7 fields of view, average of 4.33). The number of CD3+ cells in each 40X field of view were counted and the average number calculated for each tumour. To determine the ER status of the tumours, the total number of unstained and stained nuclei was counted, and amount of ER staining expressed as a percentage. Samples were considered ER+ if > 1% of tumour cells showed positive nuclear staining.
4
Immunoblot and Immunofluorescence Analysis
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Immunoblot analysis and Immunofluorescence staining were carried out with the following antibodies: anti-F4/80 (AbD Serotec), anti–ERα D6R2W (13258 Cell Signaling), and ab3575 (Abcam, Cambridge, UK), anti-Actin C-2 (sc-8432 Santa Cruz, Santa Cruz, CA, USA), and anti-GAPDH (RPCA Encor, Gainesville, FL, USA).
5
Uterus and PEC Protein Extraction and Analysis
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Total protein was extracted from the uterus and the PECs using radioimmunoprecipitation assay lysis buffer supplemented with phenylmethanesulfonyl fluoride (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions and detected by the Bicinchoninic Acid Protein Assay Kit (Tiangen Biotech Co., Ltd., Beijing, China). Approximately 50 μg of protein was separated by electrophoresis on polyacrylamide gels and subsequently transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk powder for 1.5 h and incubated overnight at 4 °C with the following primary antibodies: anti-ER-α (1:1000; ab3575, Abcam, Shanghai, China), anti- ER-β (1:1000; ab3576, Abcam), anti-PR (1:200; sc-539, Santa Cruz Biotechnology, Shanghai, China), anti-GAPDH (1:5000; Abcam) and anti-actin (1:5000; Abcam). After incubation, the membranes were washed three times with TBST and incubated with anti-rabbit/mouse IgG antibody (1:2000; CWBIO, Beijing, China) for 2 h at 37 °C, followed by washing with TBST. Finally, the membranes were immersed in a high-sensitivity luminescence reagent (BeyoECL Plus; Beyotime), exposed to film using a FusionCapt Advance FX7 (Beijing Oriental Science and Technology Development Co., Ltd., Beijing, China) and analyzed using Ipp 6.0 (Image Pro-Plus 6.0; Media Cybernetics).
6
Placental Immunohistochemical Analysis
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The formalin-fixed placenta samples underwent routine histological processing. For immunohistochemical analysis, paraffin sections (5 μm) were stained with primary antibodies to ERα (ab3575, 1:100, Abcam), CD68 (ab125212, 1:100, Abcam), CD206 (ab64693, 1:100, Abcam) manually. The immunoperoxidase-stained sections were counterstained with Mayer’s hematoxylin. Paraffin sections were stained with primary to CD56 (NCL-CD56-504, clone CD564, Leica Biosystems, Wetzlar, Germany) in a Leica BOND-MAX Automated Immunostainer with further incubation with immunoperoxidase-conjugated antibodies and counterstaining with hematoxylin included in the CD56 staining kit. For each section, 10 images were obtained on a Leica DM2500 system.
7
Antibody Validation and Metabolite Analysis
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Optima LC grade acetonitrile was purchased from Merck (Merck, Darmstadt, Germany), formic acid was purchased from Fluka (Honeywell, Morris Plains, NJ, United States), and ultrapure water was prepared using a Milli-Q purification system (Merck Millipore, Darmstadt, Germany). Primary antibodies were purchased from Abcam (Cambridge, MA, United States): anti-ERα (ab3575), anti-ERβ (ab3576), anti-Ggt1 (ab109427), and anti-Anpep (CD13; ab108310). Metabolite standards were obtained from Sigma-Aldrich (Spruce St., St Louis, MO, United States).
8
Immunohistochemistry of Estrogen Receptors in DU145 Tumors
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DU145 tumors were xed with formalin. IHC staining was performed on 4 μm sections taken from para n-embedded tumor tissues. After rehydration, the sections were incubated with a 1:200 dilution of the primary antibody, rabbit anti-estrogen receptor alpha antibody (ab3575, Abcam, Cambridge, UK) and rabbit anti-estrogen receptor β antibody (ab3576, Abcam, Cambridge, UK) at 4°C overnight. Then, the sections were incubated with a biotinylated secondary antibody (ab205718, Abcam, Cambridge, UK) and treated with Avidin-biotin-peroxidase. The 3-amino-9-ethylcarbazole substrate chromogen was used, followed by tissue counterstaining with hematoxylin. Sections were examined and photographed using a confocal microscope (Olympus, Tokyo, Japan)
9
Western Blot Analysis of Estrogen Receptors
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The membranes were incubated with 1:1000 dilutions of primary rabbit anti-estrogen receptor alpha (ab3575, Abcam, Cambridge, UK), rabbit anti-estrogen receptor β (ab3576, Abcam, Cambridge, UK) and anti-GAPDH (1:50000, 60004-1-lg, Proteintech, Rosemont, USA) antibodies at 4 °C overnight. The next day, after washing with PBS three times for 10 min each, the membranes were incubated with the secondary antibodies for 1 h at room temperature. Finally, the target proteins were visualized using a LAS4000 enhanced chemiluminescence system (GE Healthcare, Wisconsin, USA).
10
Western Blot Analysis of Estrogen Receptors
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The membranes were incubated with 1:1000 dilutions of primary rabbit anti-estrogen receptor alpha (ab3575, Abcam, Cambridge, UK), rabbit anti-estrogen receptor β (ab3576, Abcam, Cambridge, UK) and anti-GAPDH (1:50000, 60004-1-lg, Proteintech, Rosemont, USA) antibodies at 4 °C overnight. The next day, after washing with PBS three times for 10 min each, the membranes were incubated with the secondary antibodies for 1 h at room temperature. Finally, the target proteins were visualized using a LAS4000 enhanced chemiluminescence system (GE Healthcare, Wisconsin, USA).
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