Ab150083

Manufactured by Abcam

Sourced in United Kingdom, United States

Ab150083 is an Antibody to Estrogen Receptor alpha (ER alpha) from Abcam. It is a mouse monoclonal antibody targeting the estrogen receptor alpha protein.

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Lab products found in correlation

59 protocols using ab150083

Protein Localization in Transfected Cells

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The 293T cells were cultured at coverslips and transfected with RPL15-mCherry_4 and RPL-histone3.3 mRNA by TransIT, and fixed by 4% PFA for antibody immunofluorescence staining after 24 h of transfection. The samples were incubated in primary antibodies (1:500) for 3 h and secondary antibodies conjugated with Alexa Fluor 647 (1:500) for 2 h, with 15 min DAPI staining before being mounted in Prolong gold antifade reagent (ThermoFisher Scientific, P36934). For SiRNeoblasts, 20,000 cells were sorted out for each group and transfected with RPL15-mCherry_4 and RPL-histone3.3 mRNA by TransIT. At 24 hpt, following fixed by 4% FA in 0.4X PBS twice for 10 min, incubated in Hybe at 56°C for 2 h and blocked by 10% Horse serum in PBSTx0.1% at room temperature for 30 min, the cells were stained with primary antibodies (1:500) and secondary antibodies conjugated with Alexa Fluor 555 (1:500) for 2 h, respectively. The antibodies included rabbit polyclonal mCherry antibody (MBL, PM005), mouse monoclonal Flag antibody clone M2 (Sigma, F1804), goat anti-mouse IgG antibody (H + L) Alexa Fluor 647 (Abcam, ab150119), goat anti-rabbit IgG antibody (H + L) Alexa Fluor 647 (Abcam, ab150083), goat anti-mouse IgG antibody (H + L) Alexa Fluor 555 (Abcam, ab150118) and goat anti-rabbit IgG antibody (H + L) Alexa Fluor 555 (Abcam, ab150086).

Lei K., Zhang W., Chen J., McKinney S.A., Ross E.J., Lee H.C, & Sánchez Alvarado A. (2023). Pluripotency retention and exogenous mRNA introduction in planarian stem cells in culture. iScience, 26(2), 106001.

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Immunohistochemical Analysis of Murine Osteoarthritis

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Uninjured mice were sacrificed at the age of 10 weeks. In DMM or sham surgery groups, animals were sacrificed at either 2 weeks or 8 weeks postoperatively. Specimens were fixed in 4% paraformaldehyde for 24 hours, decalcified with 14% ethylenediaminetetraacetic acid (EDTA) for 14 days, and embedded in the optimum cutting temperature (OCT) compound. Sections were prepared at 6 μm thickness with a Cryofilm type 3c (SECTION-LAB, Japan).
For immunofluorescent immunohistochemistry, sections were washed in 1× PBS, blocked in 5% normal goat serum (S-1000, Vector Labs, CA, USA) for 30 min, and incubated with primary antibodies specific for CD31 (1:50, ab28364, Abcam, MA, USA), α-SMA (1:400, ab7817, Abcam), ED-A fibronectin (1:400, F6140, Sigma Aldrich, MO, USA) or TGF-β1(1:400, ab92486, Abcam) at 4 °C overnight. Sections were then incubated with Alexa Fluor® 647-conjugated secondary antibodies (1:200, ab150083 or ab150119, Abcam), and mounted with mounting medium containing DAPI (H-1500, Vector Labs). Bright field images were obtained on a Leica DM 6B microscope (Leica Biosystems, Germany). Immunofluorescent images were acquired on a LSM 780 FCS confocal microscope (Carl Zeiss, Germany).

Sono T., Hsu C.Y., Negri S., Miller S., Wang Y., Xu J., Meyers C.A., Peault B, & James A.W. (2020). Platelet Derived Growth Factor Receptor-β (PDGFRβ) lineage tracing highlights perivascular cell to myofibroblast transdifferentiation during post-traumatic osteoarthritis.. Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 38(11), 2484-2494.

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Multifunctional Hydrogel for Skin Tissue Engineering

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Polydimethylsiloxane (PDMS, Sylgard 184) was obtained from Dow Corning (Midland, USA). Polylactic acid (PLA) was purchased from Lakeshore Biomaterials Inc. (AL, USA). NaOH was purchased from Shanghai Aladdin Bio-Chem Technology Co., LTD (Shanghai, China). 1,4-butanediol diglycidyl ether (BDDE, Mw = 202.25 Da) was obtained from Adamas Reagent Co. Ltd. (Basel, Switzerland). Genipin was purchased from J&K Scientific (Beijing, China). Gelatin (Gel) from cold water fish skin and from porcine skin, hyaluronic acid (HA, Mw ≈ 20,000–400,000 Da) were purchased from Sigma-Aldrich (MO, USA). Human epidermal growth factor (EGF), FITC-labeled EGF, glutathione, DMEM high glucose medium, penicillin/streptomycin, CCK-8, Triton X-100, DAPI and BSA were purchased from Beijing Solarbio Technology Co., Ltd (Beijing, China). 1640 medium and fetal bovine serum (FBS) were obtained from Gibco. The antibodies used in this study were summarized as follows (category number, company): Phalloidin-iFluor 488 (ab176753, Abcam), anti-EGFR (ab52894, Abcam), anti-PI3 Kinase p110 beta (ab151549, Abcam), Goat Anti-Rabbit IgG H&L (Alexa Fluor 647) (ab150083, Abcam) and Goat Anti-Rabbit IgG H&L (FITC) (ab6717, Abcam).

Yang Y., Luo R., Chao S., Xue J., Jiang D., Feng Y.H., Guo X.D., Luo D., Zhang J., Li Z, & Wang Z.L. (2022). Improved pharmacodynamics of epidermal growth factor via microneedles-based self-powered transcutaneous electrical stimulation. Nature Communications, 13, 6908.

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Immunofluorescence Staining of IFITM3

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Cells were grown on glass coverslips in six-well plates. After treatments, cells were washed with PBS twice, fixed with 4% PFA for 10 min, permeabilized with 0.2% Triton X-100 for 10 min, blocked with 1% bovine serum albumin (BSA, Roche, 735094) for 1 h, and incubated with primary antibody rabbit anti-IFITM3 (1:100 diluted in 1% BSA; Proteintech group, 11714-1-AP) for 1.5 h, followed by secondary antibody anti-rabbit-Alexa Fluor 647 conjugate (1:1,000; Abcam, ab150083) for 1 h at room temperature. Finally, the coverslips were mounted onto slides using mounting medium containing DAPI, and the cells were examined by inverted LSM 780 laser scanning confocal microscope (Zeiss).

Spence J.S., He R., Hoffmann H.H., Das T., Thinon E., Rice C.M., Peng T., Chandran K, & Hang H.C. (2019). IFITM3 directly engages and shuttles incoming virus particles to lysosomes. Nature Chemical Biology, 15(3), 259-268.

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Immunofluorescence Staining of α-SMA and Desmin

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Immunofluorescence staining assays were performed as we described previously [15 (link)]. Cells were incubated overnight with the primary antibody against α-SMA (ab5694) and desmin (ab15200) (1 : 50 dilution; Abcam, USA) at 4°C. The cells were washed three times with PBS (5 min each) and then incubated in the dark for 1 h at room temperature with Alexa Fluor 488 and 550 conjugated goat anti-rat secondary antibody (1 : 200 dilution; ab150157 and ab150083, Abcam, USA). Nuclei of cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, USA). Images were obtained using a Zeiss LSM 510 META Confocal microscope using 20x/0.5 w and 40x/1.2 w objectives.

Zhang Q., Xiang S., Liu Q., Gu T., Yao Y, & Lu X. (2018). PPARγ Antagonizes Hypoxia-Induced Activation of Hepatic Stellate Cell through Cross Mediating PI3K/AKT and cGMP/PKG Signaling. PPAR Research, 2018, 6970407.

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Immunofluorescence Staining of PRPF3 and TMEM43

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Tumor cells were washed twice with PBS (HyClone, Utah, USA), and fixed using 4% paraformaldehyde (Biosharp, Shanghai, China) for 15 min. Then, 0.3% Triton X-100 was used for permeation for 5 min and 5% BSA was used to block for 45 min. The following primary antibodies were used: rabbit anti-PRPF3 (A5482, Proteintech, China) and mouse anti-TMEM43 (SC-365298, Santa, USA), followed by the secondary antibodies Alexa Fluor 647-labeled anti-rabbit (ab150083, abcam, USA) and FITC-labeled anti-mouse (ab6785, abcam, USA). Cell images were captured on a Nikon Eclipse Ti-SR system.

Li J., Song Y., Zhang C., Wang R., Hua L., Guo Y., Gan D., Zhu L., Li S., Ma P., Yang C., Li H., Yang J., Shi J., Liu X, & Su H. (2022). TMEM43 promotes pancreatic cancer progression by stabilizing PRPF3 and regulating RAP2B/ERK axis. Cellular & Molecular Biology Letters, 27, 24.

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Immunostaining of Ischemic Rat Brain Regions

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As previously described (Geng et al., 2021 (link)), the frozen rat brain sections were prepared and the ischemic MCA penumbra region was observed. For immunofluorescence, brain sections were incubated with a mixture of PCK1 (Proteintech Cat# 16754‐1‐AP, RRID:AB_2160031), PCK2 (Proteintech Cat# 14892‐1‐AP, RRID:AB_2160044) or p‐FoxO1 (1:100, CST, USA) and neuronal specific nuclear protein (NeuN) (Abcam Cat# ab104224, RRID:AB_10711040), followed by a mixture of Alexa Fluor 488 or 647 fluorescence tagged secondary antibodies (Abcam Cat# ab150113, RRID:AB_2576208;Abcam Cat# ab150083, RRID:AB_2714032). The images were observed by using a fluorescence microscope and a Leica TCS‐SP5 confocal microscope.

Li F., Geng X., Ilagan R., Bai S., Chen Y, & Ding Y. (2022). Exercise postconditioning reduces ischemic injury via suppression of cerebral gluconeogenesis in rats. Brain and Behavior, 13(1), e2805.

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Histological Analysis of Murine Knee Joint

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Stifle joints were fixed in 4% paraformaldehyde for 24 h, decalcified with 14% ethylenediaminetetraacetic acid (EDTA) for 14 d, and embedded in OCT compound (Sakura, Torrance, California).Sagittal sections of the stifle joint were prepared at 6 µm thickness with a Cryofilm type 3c (SECTION-LAB, Hiroshima, Japan). Inguinal fat pads from donor Pdgfra orPdgfrβ‐CreER^T2‐mT/mG mice were also dissected, embedded in OCT, and cryosectioned at 30 μm thickness. Routine safranin O / fast greenstaining of stifle joints section was performed with methods adopted from past work^{25 (link)–27 (link)}.
For immunofluorescent immunohistochemistry, sections were washed in 1× PBS, blocked in 5% normal goat serum (S-1000, Vector Labs, CA, USA) for 30 min, and incubated with primary antibodies specific for ACAN (1:100, ab3778, Abcam, MA, USA), COL10(1:100, ab58632, Abcam), CD31 (1:100, ab 28364, Abcam)and scleraxis (1:100, ab58655, Abcam) at 4 °C overnight. Sections were then incubated with Alexa Fluor® 647-conjugated secondary antibodies (1:200, ab150083, Abcam) or DyLight® 594 (1:100, DI-1594, Vector Labs), and mounted with mounting medium containing DAPI (H-1500, Vector Labs, Burlingame, CA)(Supplemental Table 1). Immunofluorescence images were acquired on a Leica DM 6B or LSM 880 FCS confocal microscope (Carl Zeiss, Oberkochen, Germany).

Hsu G.C., Cherief M., Sono T., Wang Y., Negri S., Xu J., Peault B, & James A.W. (2021). Divergent effects of distinct perivascular cell subsets for intra-articular cell therapy in post-traumatic osteoarthritis. Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 39(11), 2388-2397.

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Quantifying Wound Immune Cells

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Wound beds were surgically removed at WD2 and WD7, using a 6‐mm biopsy punch to remove excess tissue. Cell suspensions were prepared by digesting the tissue in a cocktail consisting of Liberase TL (Roche, 5401020001) and DNase I (Sigma, DN25) in RPMI 1640 (Gibco, 11875093). Cells were washed and then Fc blocked (BioLegend, 101320), before staining with an antibody cocktail containing anti‐MPO (Abcam, ab208670, 1:500) or respective isotype control (Abcam, ab172730, 1:500). Cells were then stained with a secondary Alexa Fluor 647 antibody (Abcam, ab150083, 1:2000). On the final wash, SYTOX green was added (Thermo Fisher, S7020, 1:1000). This was performed on a BD LSR II, and downstream analysis of data was performed using FlowJo.

Wier E., Asada M., Wang G., Alphonse M.P., Li A., Hintelmann C., Sweren E., Youn C., Pielstick B., Ortines R., Lyu C., Daskam M., Miller L.S., Archer N.K, & Garza L.A. (2021). Neutrophil extracellular traps impair regeneration. Journal of Cellular and Molecular Medicine, 25(21), 10008-10019.

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Immunofluorescence Assay for LC3B

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Cells of each group were inoculated in petri dishes with coverslip to prepare cell slides, which were fixed in 4% paraformaldehyde for 30 min, cleaned with PB saline (PBS) for 3 times, and penetrated in 0.2% Triton X-100 for 3 min. Then, the cell slide was cleaned with PBS for 3 times, and blocked with goat serum at 25°C for 30 min. The anti-LC3B (ab192890, 1:200, Abcam, UK) was appended and placed at 4°C for 12 h and then the cell slide was cleaned with PBS for 3 times. The cell slide was further mixed with the secondary antibody (ab150083, 1:1000, Abcam) in the dark for 2 h. After cleaned in PBS for 3 times, the cell slide was dyed with DAPI, and then photographed under an AX-70 fluorescent microscopy (Leica Microsystems Inc.).

Guo F., Wen W., Mi Z., Long C., Shi Q., Yang M., Zhao J, & Ma R. (2024). NRSN2 promotes the malignant behavior of HPV-transfected laryngeal carcinoma cells through AMPK/ULK1 pathway mediated autophagy activation. Cancer Biology & Therapy, 25(1), 2334463.

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