Gfra1

Manufactured by Santa Cruz Biotechnology

Sourced in United Kingdom

GFRA1 is a protein that functions as a receptor for the GDNF (Glial Cell Derived Neurotrophic Factor) ligand. It plays a role in the survival and differentiation of certain types of neurons.

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Lab products found in correlation

Uchl1, by Bio-Rad (6 mentions) Gpr125, by Abcam (4 mentions) Fluorescence microscope, by Leica (3 mentions) Triton x 100, by Merck Group (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Alexa fluor 594, by Thermo Fisher Scientific (2 mentions) Triton x, by Merck Group (1 mentions) Nanog, by Cell Signaling Technology (1 mentions) Ssea1, by BD (1 mentions) Proliferating cell nuclear antigen (pcna), by Abcam (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Fitc conjugated or rhodamine conjugated igg secondary antibodies, by Merck Group (1 mentions) Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (1 mentions) Ab133350, by Santa Cruz Biotechnology (1 mentions) Stem cell factor (scf), by Merck Group (1 mentions) Bone morphogenetic protein 4 (bmp4), by Abcam (1 mentions) Piwil1, by Abcam (1 mentions) Piwil2, by Abcam (1 mentions) Acrosin, by Santa Cruz Biotechnology (1 mentions) γh2ax, by Merck Group (1 mentions)

8 protocols using gfra1

Immunofluorescence Staining of Germ Cells

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The cells grown on glass slips were fixed with 4% PFA in PBS. After 3 washes with PBS, they were permeabilized in 0.1% Triton X (Sigma) and then blocked with 10% normal donkey serum. Cells were then incubated overnight at 4 °C with the primary antibodies. After washing with PBS for three times, cells were then incubated by the corresponding conjugated secondary antibodies, Alexa Fluor 488, Alexa Fluor 594, and Alexa Fluor 647 at 400-fold dilution in 3% BSA in PBS. Cell nuclei were labeled with DAPI (Sigma). The imagines were visualized by using an inverted fluorescence microscope or ZEISS confocal microscope.
The primary antibodies were shown as follows: PLZF (1:100, mouse, Abcam), UCHL1 (1:200, mouse, AbD Serotec), CD90 (1:100, rabbit, Abcam), GPR125 (1:100, rabbit, Abcam), GFRΑ1 (1:150, rabbit, Sigma), GFRA1 (1:50, mouse, Santa Cruz), MAGEA (1:100, rabbit, Abcam), SCP3 (1:100, rabbit, Abcam), SSEA1 (1:100, mouse, BD), c-kit (1:100, rabbit, Abcam), VASA (DDX4, 1:200, rabbit, Abcam), PCNA (1:100, rabbit, Abcam), OCT4 (1:200, rabbit, a gift from Ying Jin), Nanog (1:100, Cell Signaling Technology), and SOX2 (1:100, rabbit, Abcam).

Xu H., Yang M., Tian R., Wang Y., Liu L., Zhu Z., Yang S., Yuan Q., Niu M., Yao C., Zhi E., Li P., Zhou C., He Z., Li Z, & Gao W.Q. (2020). Derivation and propagation of spermatogonial stem cells from human pluripotent cells. Stem Cell Research & Therapy, 11, 408.

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Immortalized Human Male Germline Stem Cells Characterization

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The phenotypic characteristics of the immortalized human male germline stem cells were identified by immunocytochemistry according to the procedure described previously46 (link). The cultured cells or the cells using cytospin were fixed with 4% paraformaldehyde (PFA) and permeabilized in 0.4% triton-X 100 (Sigma-Aldrich) for 15 min at room temperature and followed by three washes in phosphate-buffered saline (PBS) for 5 min each. After blocking in 1% bovine serum albumin for 1 hour, the cells were incubated with primary antibodies, including SV40 (Santa Cruz), GFP (Abcam), DAZL (Abcam), VASA (Abcam), RET (Santa Cruz), UCHL1 (AbD Serotec), PLZF (Abcam), GFRA1 (Santa Cruz), PCNA (Santa Cruz), GPR125 (Abcam), and THY1 (Abcam) at a dilution with 1:200 overnight at 4 °C. After washes three times in PBS for 5 min each, the cells were incubated with appropriate FITC-conjugated or rhodamine-conjugated IgG secondary antibodies (Sigma) for 1 hour at room temperature. The nuclei of cells were stained by DAPI (4′ -6-diamidino-2-phenylindole) and the cells were observed the epifluorescence using fluorescence microscope (Leica).

Hou J., Niu M., Liu L., Zhu Z., Wang X., Sun M., Yuan Q., Yang S., Zeng W., Liu Y., Li Z, & He Z. (2015). Establishment and Characterization of Human Germline Stem Cell Line with Unlimited Proliferation Potentials and no Tumor Formation. Scientific Reports, 5, 16922.

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Immunofluorescent Characterization of Spermatogenic Cells

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The freshly isolated human spermatogonia and round spermatids were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.5% Triton X-100 for 30 min, blocked with 5% BSA for 1 hr, and incubated with primary antibodies. The primary antibodies included THY1 (Abcam, ab133350, 1:200), GFRA1 (Santa Cruz, sc-6156, 1:200), PLZF (Santa Cruz, sc-22839, 1:200), UCHL1 (Bio-Rad, MCA4750, 1:200), PNA (Life Technologies, L32458, 1:200), and Protamine 2 (PRM2) (Atlas Antibodies, HPA056386, 1:200). After incubation at 4°C overnight, cells were washed three times in PBS (Medicago, Uppsala, Sweden), followed by incubation with secondary antibodies for 1 hr at room temperature. Secondary antibodies were conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen). DAPI was used to label cell nuclei, and images were captured with a fluorescence microscope (Leica). Isotype IgGs replaced primary antibodies and served as negative controls.

Yao C., Yuan Q., Niu M., Fu H., Zhou F., Zhang W., Wang H., Wen L., Wu L., Li Z, & He Z. (2017). Distinct Expression Profiles and Novel Targets of MicroRNAs in Human Spermatogonia, Pachytene Spermatocytes, and Round Spermatids between OA Patients and NOA Patients. Molecular Therapy. Nucleic Acids, 9, 182-194.

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Multiplex Immunostaining of Testicular Cells

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Cells were fixed in 4% PFA for 15 min at room temperature, permeabilized with 0.5% Triton X-100 for 30 min, blocked with 5% BSA for 1 h, and incubated with primary antibodies. The primary antibodies included GPR125 (Santa Cruz,1:200), GFRA1 (Santa Cruz,1:200), UCHL1 (AbD Serotec,1:200), PLZF (Abcam, 1:200), MAGEA4 (a kind gift from Professor Giulio C. Spagnoli, University Hospital of Basel, Switzerland, 1:25), SOX9 (Millipore, 1:200), WT1 (Santa Cruz, 1:200), SCF (Sigma, 1:200), BMP4 (Abcam, 1:200), PIWIL1 (Abcam, 1:200), PIWIL2 (Abcam, 1:200), γH2AX (Millipore, 1:100), Protamine 2 (PRM2, Santa Cruz, 1:200), Acrosin (ACR, Santa Cruz, 1:200), SYCP3 (Abcam, 1:100), H3K9 trimethylation (H3K9me3, Abcam, 1:200), and Human nuclear antigen (HumNuc, Millipore, 1:200). After incubation at 4 °C overnight, cells were washed three times in PBS (Sigma) and followed by incubation with secondary antibodies for 1 h at room temperature. Secondary antibodies were conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen). DAPI was used to label cell nuclei, and images were captured with a fluorescence microscope (Leica). Replacement of primary antibodies with isotype IgGs served as negative controls.

Sun M., Yuan Q., Niu M., Wang H., Wen L., Yao C., Hou J., Chen Z., Fu H., Zhou F., Li C., Gao S., Gao W.Q., Li Z, & He Z. (2018). Efficient generation of functional haploid spermatids from human germline stem cells by three-dimensional-induced system. Cell Death and Differentiation, 25(4), 747-764.

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Isolation and Characterization of Human Sertoli Cells

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Human Sertoli cells were isolated from obstructive azoospermia patients with normal spermatogenesis using a two-step enzymatic digestion and followed by differential plating. Immunocytochemistry was performed to determine the identity of human SSC line, primary human Sertoli cells, and mouse liver mesenchymal cells pursuant to the procedure described previously [37 ]. Briefly, cells were fixed in 4% paraformaldehyde (PFA) for 30 min and followed by permeabilization with 0.4% Triton X-100 for 15 min and blocking in 1% bovine serum albumin (BSA, Sigma-Aldrich) for 1 h. The primary antibodies were as follows: UCHL1 (AbD Serotec), GFRA1 (Santa Cruz), GPR125 (Abcam), PLZF (Santa Cruz), VIMENTIN (Abcam), and VWF (Santa Cruz). After incubation overnight at 4°C, rhodamine-conjugated or FITC-conjugated IgG was used as secondary antibodies. The dilutions, resources, and companies of primary and secondary antibodies for immunocytochemistry were shown in Table 2. Double immunostaining was also performed to verify the identity of human SSC line antibodies against GFRA1 and UCHL1 as well as GPR125 and UCHL1. Replacement of primary antibodies with isotype IgG or PBS served as a negative control. The nuclei of cells were stained with DAPI (4′-6-diamidino-2-phenylindole) and the epifluorescence were examined under fluorescence microscope (Nikon Eclipse Ti-S, Nikon Corporation, Tokyo, Japan).

Chen Z., Niu M., Sun M., Yuan Q., Yao C., Hou J., Wang H., Wen L., Fu H., Zhou F., Li Z, & He Z. (2017). Transdifferentiation of human male germline stem cells to hepatocytes in vivo via the transplantation under renal capsules. Oncotarget, 8(9), 14576-14592.

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Immunofluorescence Staining of Human Stem Cells

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Immunofluorescence staining was performed to determine the identity of cultured hSSCs and the differentiated hSSCs by the induction system pursuant to the procedure as described previously [27 (link)]. Briefly, cells on coverslips or plates were fixed in 4% paraformaldehyde for 20 min, permeabilized with or without 0.01% Triton X-100 (Sigma) for 10 min, and blocked in 4% normal donkey or goat serum at room temperature. Concomitantly, cells were incubated with primary antibodies at a dilution of 1:200–1000, respectively, overnight at 4 °C. The primary antibodies used in this study were as follows: UCHL1 (AbDSerotec), GFRA1, EN-1, OTX2 (Santa Cruz), GPR125, Tuj-1, PLZF, FoxA2 Lmx1a, Lmx1b (Abcam), DAT (Millipore), Curr1 (Millipore), PitX3 (GeneTex), and SYN (Chemicon). After three washes in PBS, the cells were incubated with corresponding Alexa Fluor® 488, Alexa Fluor® 594, or FITC-conjugated-IgG secondary antibodies at a 1:200–1:500 dilution for 1 h at RT. DAPI (4″-6-diamidino-2-phenylindole) was used to stain cell nuclei, and the cells were observed under a fluorescence microscope (Olympus BX-53, Japan).

Yang H., Hao D., Liu C., Huang D., Chen B., Fan H., Liu C., Zhang L., Zhang Q., An J, & Zhao J. (2019). Generation of functional dopaminergic neurons from human spermatogonial stem cells to rescue parkinsonian phenotypes. Stem Cell Research & Therapy, 10, 195.

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Immunofluorescence Staining of GFRA1, SETDB1, H3K9me3, and c-KIT

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The procedure was similar to that in immunohistochemistry section with minor modification. Briefly, the slides were incubated with 10% donkey serum for blocking nonspecific reactions at room temperature for 2 h and incubated with the primary antibodies against GFRA1 (1:50; Santa Cruz Biotechnology), SETDB1 (1:50; Santa Cruz Biotechnology), H3K9me3 (1:50; Millipore) and c-KIT (1:50; Santa Cruz Biotechnology) respectively, overnight at 4°C. Next day, the sections were washed four times with PBS and then incubated with either donkey anti-Goat (1:200; Abcam) or donkey anti-Rabbit (1:100; Santa Cruz Biotechnology) antibodies for immunofluorescence labeling. Slides were washed and counterstained with DAPI (CWBIO). Fluorescent images were captured with the Nikon Eclipse 80i fluorescence microscope camera.

Liu T., Zhang P., Li T., Chen X., Zhu Z., Lyu Y., Li X., Tian X, & Zeng W. (2017). SETDB1 plays an essential role in maintenance of gonocyte survival in pigs. Reproduction (Cambridge, England), 154(1).

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Protein Analysis of Embryoid Bodies

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The EBs derived from iPS cells were lysed using the RIPA buffer (Santa Cruz) containing a cocktail of protease inhibitors (Roche). Cell lysates were cleared by centrifugation at 12,000 g, and the concentrations of total proteins were measured by BCA kit (Dingguo Company, Beijing, China). Twenty micrograms of total protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. After blocking with 5% BSA and 0.1% Tween20 for 1 h at room temperature, membranes were probed with primary antibodies, including PRDM1 (Abnova), VASA (Abcam), DAZL (Abcam), UCHL1 (AbD Serotec Kidlington, UK), GFRA1 (Santa Cruz), KIT (Abcam), phospho-Smad1/5 (Cell signaling), Smad5 (Cell signaling) and ACTB (beta-actin, Proteintech) overnight at 4°C. The detailed information on antibodies was shown in Table 3. The blots were incubated with HRP-conjugated antirabbit or anti-goat IgG polyclonal secondary antibodies (Santa Cruz) at 1:2500 dilution for 1 h at room temperature. After extensive washes with TBST, the blots were visualized using an enhanced-chemiluminescent detection kit (Santa Cruz).

Yang S., Yuan Q., Niu M., Hou J., Zhu Z., Sun M., Li Z, & He Z. (2017). BMP4 promotes mouse iPS cell differentiation to male germ cells via Smad1/5, Gata4, Id1 and Id2. Reproduction (Cambridge, England), 153(2).

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