Anti scf

Manufactured by Merck Group

Sourced in United States

Anti-SCF is a laboratory reagent produced by Merck Group. It is a monoclonal antibody that binds to the stem cell factor (SCF) protein, also known as Kit ligand. The core function of Anti-SCF is to facilitate the study and analysis of SCF and its role in cellular processes.

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5 protocols using anti scf

Immunocytochemical Analysis of Sertoli Cells

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For immunocytochemical staining, the immortalized human Sertoli cells were fixed with 4% paraformaldehyde (PFA) for 15 min, washed three times with phosphate-buffered saline (PBS) for 5 min each and permeabilized in 0.5% triton-X 100 (Sigma) for 20 min. After washing with PBS, the cells were blocked in 3% serum or BSA for 60 min and followed by incubation with primary antibodies, including anti-SOX9 (Millipore), anti-WT1 (Santa Cruz), anti-OCLN (Abcam), anti-VIM (vimentin, Santa Cruz), anti-SCF (Sigma), anti-BMP4 (Abcam), anti-GDNF (Abcam), anti-3β-HSD (Santa Cruz), anti-VASA(Santa Cruz), and anti-Ki-67 (BD Biosciences), overnight at 4°C. Replacement of primary antibodies with isotype IgGs (Santa Cruz) served as negative controls. After washing three times in PBS for 10 min each, the cells were incubated with the secondary antibody (Sigma) conjugated with rhodamine at a 1:200 dilution for 1 hour at room temperature. DAPI (4, 6-diamidino-2-phenylindole, Sigma) was used to counterstain the nuclei, and images were captured with a fluorescence microscope (Nikon).

Wen L., Yuan Q., Sun M., Niu M., Wang H., Fu H., Zhou F., Yao C., Wang X., Li Z, & He Z. (2017). Generation and characteristics of human Sertoli cell line immortalized by overexpression of human telomerase. Oncotarget, 8(10), 16553-16570.

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Sertoli Cell Protein Expression Analysis

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The cultured human Sertoli cells at different passages were lysed with RIPA buffer (Santa Cruz) for 30 min on ice. After 30 min lysis on ice, cell lysates were cleared by centrifugation at 12,000 g, and the concentration of protein was measured by BCA kit (Dingguo Company, China). Thirty micrograms of cell lysate from each sample were used for SDS-PAGE (Bio-Rad Laboratories, Richmond, CA), and Western blots were performed according to the protocol as described previously [11 (link)]. The chosen antibodies included anti-SCF (Sigma), anti-GDNF (Santa Cruz), anti-BMP4 (Abcam), anti-PCNA (Santa Cruz), anti-phos-AKT (Cell Signaling), and anti-phos-SMAD1/5 (Cell Signaling). After extensive washes with PBS, the blots were detected by chemiluminescence (Chemi-Doc XRS, Bio-Rad), and the integrated density values (IDV) was calculated by comparing the signals of target proteins with that of housekeeper ACTB (Santa Cruz).

Guo Y., Hai Y., Yao C., Chen Z., Hou J., Li Z, & He Z. (2015). Long-term culture and significant expansion of human Sertoli cells whilst maintaining stable global phenotype and AKT and SMAD1/5 activation. Cell Communication and Signaling : CCS, 13, 20.

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Evaluating c-kit and SCF Levels in Colon

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Protein contents of c-kit and SCF in colon samples homogenates were evaluated by Western blot. In brief, the total proteins of homogenates were determined using a bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL, USA). Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) was used to separate proteins in BioRad electrophoresis system (BioRad Laboratories, Hercules, CA, USA). Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes. After 2 h treatment with blocking buffer in TBS containing 5% nonfat milk at room temperature, PVDF membranes were incubated overnight with 1 : 1000 diluted anti-c-kit (Santa Cruz, CA, USA), 1 : 500 diluted anti-SCF (Sigma, MO, USA), and 1 : 8000 diluted anti-β-actin (Kangchen, Shanghai, China) antibodies, respectively. Binding of the primary antibody was detected using the corresponding HRP-conjugated secondary antibody (Beyotime, Jiangsu, China). An enhanced chemiluminescence kit (Pierce Biotechnology Inc., Rockford, IL, USA) and the Odyssey Infrared Imaging System (Gene Company Ltd., Hong Kong) were used for chemiluminescence detection. Housekeeping protein β-actin was used as a loading control. The amount of protein expression is presented relative to the levels of β-actin.

Yan S., Yue Y.Z., Wang X.P., Dong H.L., Zhen S.G., Wu B.S, & Qian H.H. (2017). Aqueous Extracts of Herba Cistanche Promoted Intestinal Motility in Loperamide-Induced Constipation Rats by Ameliorating the Interstitial Cells of Cajal. Evidence-based Complementary and Alternative Medicine : eCAM, 2017, 6236904.

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Immunocytochemical Analysis of Human Sertoli and Germ Cells

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For immunocytochemical staining, the freshly isolated and cultured human Sertoli cells as well as human male germ cells were fixed with 4% paraformaldehyde (PFA) for 30 min, washed three times with cold phosphate-buffered saline (PBS) and permeabilized in 0.4% triton-X 100 (Sigma) for 5 min. After washing with PBS, the cells were blocked in 2% BSA for 30 min and followed by incubation with primary antibodies, including anti-GATA4 (Santa Cruz), WT1 (Santa Cruz), GDNF (Santa Cruz), anti-SCF (Sigma), anti-BMP4 (Abcam), anti- VIM (Santa Cruz), anti-PCNA (Sigma), anti-SMA (smooth muscle alpha actin, Abcam) and CYP11A1 (cholesterol side-chain cleavage enzyme, Abcam) at a dilution with 1:200 overnight at 4°C. Isotype IgGs for the first antibody were used as the negative controls. After extensive washes with PBS for 30 min, the cells were incubated with the secondary antibody IgG (Sigma) conjugated with fluorescein isothiocyanate (FITC) or rhodamine at a 1:200 dilution for 1 hour at room temperature. DAPI (4,6-diamidino-2-phenylindole) was used to label the nuclei, and the cells were observed for epifluorescence under a fluorescence microscope (Leica).

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Immunocytochemical Analysis of Sertoli Cell Markers

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For immunocytochemical staining, human Sertoli cells were fixed with 4% paraformaldehyde (PFA) for 30 min, washed three times with cold phosphate-buffered saline (PBS) and permeabilized in 0.4% triton-X 100 (Sigma) for 5 min. After washing with PBS, the cells were blocked in 2% BSA for 30 min and followed by incubation with primary antibodies, including anti-GATA4 (Santa Cruz, catalog no: SC-1237, dilution: 1:200), anti-WT1 (Santa Cruz, catalog no: SC-192, dilution: 1:200), anti-SOX9 (Millipore, catalog no: AB5535, dilution: 1:100), anti-GDNF (Santa Cruz, catalog no: SC-328, dilution: 1:200), anti-SCF (Sigma, catalog no: SAB3500292, dilution: 1:200), anti-BMP4 (Abcam, catalog no: ab124715, dilution: 1:200), anti-CYP11A1 (Abcam, catalog no: ab75479, dilution: 1:200), and anti-smooth muscle actin (SMA, Abcam, catalog no: ab5694, dilution: 1:200 ) overnight at 4°C. Replacement of primary antibodies with isotype IgGs (Santa Cruz, at 1:50 dilutions) served as negative controls. After extensive washes with PBS for 30 min, the cells were incubated with the secondary antibody IgG (Sigma) conjugated with fluorescein isothiocyanate (FITC) or rhodamine at a 1:200 dilution for 1 hour at room temperature. DAPI (4, 6-diamidino-2-phenylindole) was used to label the nuclei, and images were captured with a fluorescence microscope (Nikon).

Yao C., Sun M., Yuan Q., Niu M., Chen Z., Hou J., Wang H., Wen L., Liu Y., Li Z, & He Z. (2016). MiRNA-133b promotes the proliferation of human Sertoli cells through targeting GLI3. Oncotarget, 7(3), 2201-2219.

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