Immunostaining of the OE was performed on 12 μm coronal cryosections of paraformaldehyde-fixed tissue from adult C57BL6 mice on superfrost slides (Thermo Scientific, Menzel Gläser). After blocking with 1% fish gelatin in phosphate-buffered saline containing 0.1% Triton X-100, sections were incubated with primary antibody (anti-Paqr8 (Abcam): 1:100, anti-Pacr9 (Abcam): 1:50, anti-GPR30 (Abcam): 1:100, anti-Pgrmc1 (Sigma-Aldrich): 1:100) and fluorescently-labeled secondary antibody (Invitrogen, anti rabbit Alexa 456 nm, 1:100) dilutions in blocking solution. Stained sections were mounted in ProLong Antifade Gold medium (Molecular Probes). All fluorescence images were collected on a confocal laser scanning microscope (LSM510 Meta, Zeiss, Oberkochen, Germany). For the anti-Pgrmc1 antibody, the staining was performed on tissue sections following a heat-induced epitope antigen retrieval using citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0). Control experiments completed without addition of the primary antibody revealed a low level of background staining.
Anti gpr30
Manufactured by Abcam
Sourced in United Kingdom
Anti-GPR30 is a primary antibody that specifically binds to the G Protein-Coupled Receptor 30 (GPR30) protein. GPR30 is a membrane-bound receptor that plays a role in estrogen signaling. This antibody can be used to detect and study the expression and localization of GPR30 in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510 meta, by Zeiss (2 mentions)
Superfrost slide, by Thermo Fisher Scientific (1 mentions)
Anti pgrmc1, by Merck Group (1 mentions)
Prolong gold antifade medium, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000 mini biomolecular imager, by GE Healthcare (1 mentions)
Phospho mek, by Cell Signaling Technology (1 mentions)
Ez ecl, by Sartorius (1 mentions)
P44 42 mapk erk1 2, by Cell Signaling Technology (1 mentions)
P fgfr1, by Abcam (1 mentions)
Phospho p44 42 mapk p erk1 2, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Gapdh, by Proteintech (1 mentions)
Ab39742, by Abcam (1 mentions)
Ab56889, by Abcam (1 mentions)
Ab6789, by Abcam (1 mentions)
Sc 166333, by Santa Cruz Biotechnology (1 mentions)
Sc 166572, by Santa Cruz Biotechnology (1 mentions)
Anti erβ, by Abcam (1 mentions)
Sc 293182, by Santa Cruz Biotechnology (1 mentions)
6 protocols using anti gpr30
1
Immunostaining of Mouse Olfactory Epithelium
2
Western Blot Analysis of FGF2, GPR30, and MAPK Signaling
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Samples were homogenized in RIPA buffer (Beyotime Biotechnology) with protease and phosphatase Inhibitor (Thermo Fisher). Western blot analyses were carried out in accordance with standard protocols, and membranes were incubated with anti-FGF2 (1:200, Proteintech), anti-GPR30 (1:100, Abcam), phospho-Mek (1:1000, Cell Signaling Technology), Mek (1:1000, Cell Signaling Technology), phospho-p44/42 MAPK (p-Erk1/2, 1:1000, Cell Signaling Technology), p44/42 MAPK (Erk1/2, 1:1000, Cell Signaling Technology), p-FGFR1 (1:500, Abcam), and GAPDH (1:5000, Proteintech). The membranes were incubated with HRP-conjugated antibodies goat anti-rabbit (1:10000, Abcam) or goat anti-mouse (1:10000, Abcam). They were imaged on the Image Quant LAS 4000 mini biomolecular imager (GE Healthcare Life Sciences) after being applied with EZ-ECL (Biological Industries). The relative protein levels were measured by Image J software (National Institutes of Health) and normalized over the expression of GAPDH.
3
GPR30 Immunofluorescence Protocol
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Cells were fixed with 4% paraformaldehyde, washed with PBS, and permeabilized with 0.1% Triton X-100 for 5 minutes. Samples were then blocked for 1 hour with 5% bovine serum albumin in PBS. Then, cells were incubated with rabbit anti-GPR30 (Abcam, Cambridge, MA) primary antibody for 1 hour at room temperature. Specific secondary antibodies conjugated with green fluorescent protein fluorochrome (Invitrogen) were subsequently incubated with cells for 1 hour at room temperature. After PBS washes, slides were mounted. Images were captured using an optical microscope (DP73, Olympus Corp.).
4
DMSO-based Cell Culture Protocol
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Dimethyl sulfoxide (DMSO, A3672) was purchased from AppliChem GmbH (Darmstadt, Germany). TRIzol reagent and reverse transcription polymerase chain reaction polymer kit (AQ131-02) were purchased from Beijing Full-Type Gold Biotechnology Co., Ltd. (Beijing, China); besides, DMEM/F12 (11330-032), antibiotics-antimycotic (15240-062), fetal bovine serum (FBS; 10099141), 0.25% trypsin-EDTA digestive juices (25200-056) were purchased from Gibco (New York, NY, USA). The cell counting kit-8 (CCK-8) was provided by Dojindo Molecular Technologies, Inc. (Rockville, MD USA). Mfn2 monoclonal antibody (dilution, 1/1000; Ab56889) was purchased from Abcam (Cambridge, UK). Anti-procollagen 1A1/1A2/3A1 antibodies (dilution, 1/200; Sc-293182, Sc-166572, and Sc-166333, respectively) were purchased from Santa Cruz Biotechnology Inc. (Dallas, Texas, USA). Anti-ERα (D8H8) Rabbit mAb antibody(dilution, 1/1000) and Anti-cyclinD1 (92G2) Rabbit mAb antibody(dilution, 1/1000) were purchased from Cell Signaling Technology, Inc. (USA). Horseradish peroxidase (HRP) goat anti-mouse IgG (dilution, 1/5000, ab6789), Anti-ERβ(dilution, 1/1000, ab196787) and Anti-GPR30 (dilution, 1/1000, ab39742) were purchased from Abcam (Cambridge, UK). Procedural cooling box was purchased from Sigma-Aldrich (St. Louis, MO, USA).
5
Immunofluorescent Analysis of hfHypo Cells
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The behavior of hfHypo cells was analyzed by Laser Scanning Confocal Microscopy (LSCM, Fluoview FV300, Olympus) after the immunostaining of neuronal markers (β-Tub III and Neuronal nuclei, NeuN), GnRH phenotype markers (GnRH peptide and kisspeptin receptor, KISS1R) and estrogenic receptors (ER β and G proteincoupled receptor 30, GPR30). Neurons were fixed in 4% (wt/vol) paraformaldehyde for 30 min, followed by permeabilization and blocking with a solution containing 0.3% (vol/vol) Triton X-100 and 10% (vol/vol) FBS in PBS for 1 h at 37 C. Samples were then incubated overnight at 4 C with the following primary antibodies: anti-βTub III (1:500, Covance), anti-NeuN (1:200, Millipore), anti-GnRH (1:100, Abcam), anti-KISS1R (1:200, Santa Cruz Biotechnology), anti-ERβ (1:200, Santa Cruz Biotechnology) and anti-GPR30 (1:200, Abcam). Secondary antibodies, Cy2 TM -conjugated Affini Pure donkey anti-rabbit IgG, Cy3 TM -conjugated Affini Pure donkey anti-mouseIgG and a Cy5 TM -conjugated Affini Pure donkey anti-goat IgG (1:500, Jackson ImmunoResearch Europe Ltd.) were then added for 1 h at RT. Finally, cells were counterstained with 200 ng/ml DAPI (Molecular Probes), for nuclear localization.
6
Western Blotting of Cell Signaling
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Western blotting was performed according to the approach previously established in our laboratory [21, 23] . After treatment, proteins were extracted from HTR8/SVneo cells for Western blotting. The primary antibodies used were anti-GPR30 (1:2000, Abcam, ab154069), anti-MMP9 (1:1000, CST, #3852), anti-p-PI3K (1:1000, CST, #4228), anti-PI3K (1:1000, CST, #5405), anti-p-Akt (1:1000, CST, #9271), anti-Akt (1:1000, CST, #9272) and anti-β-actin (1:1000, ZSGB-BIO, TA-09).
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