Ab64677

Manufactured by Abcam

Sourced in United States, United Kingdom

Ab64677 is a lab equipment product. It is a self-contained unit designed for specific laboratory processes. The core function of this product is to perform a specific task in the laboratory setting.

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Lab products found in correlation

8 protocols using ab64677

Immunohistochemical Analysis of Embryonic Development

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Embryos were fixed in 4% paraformaldehyde at 4 °C overnight (P-Smad staining) or for 45 mn (BMP4 and Noggin stainings) and embedded in OCT by flash freezing on dry ice. Transverse sections (7 μM) were produced using a CM1900 Crystat (Leica). Sections were permeabilized in PBS/0.1% Triton/1% BSA for 1 h, before blocking for 30 mn in PBS/10% fetal calf heat-inactivated serum. Immunostainings were performed using rabbit anti-P-Smad1,5,8 (1:100, clone D5B10, Cell Signaling Technology), goat anti-BMP4 (1:50, clone N-16, Santa Cruz Biotechnology), goat anti-Noggin (1:100, clone AF 719, R&D systems), rabbit anti-SCF (1:50, ab64677, Abcam), unconjugated rat anti-CD31 or PE-conjugated anti-CD31 (1:50, MEC13.3, Pharmingen), goat anti-CD45 (1:100, clone AF 114, R&D systems) and rat anti-cKit (1:100, clone 2B8, Biolegend), followed by incubation with anti-goat Alexa fluor 488 (1:100, Invitrogen), anti-goat NL577 (1:100, R&D systems), anti-rabbit Alexa fluor 488 (1:100, Invitrogen) and anti-rat Alexa fluor 647 (1:100, Invitrogen). Images were acquired with an inverted confocal microscope (SP8) and processed using FiJi software.

Souilhol C., Gonneau C., Lendinez J.G., Batsivari A., Rybtsov S., Wilson H., Morgado-Palacin L., Hills D., Taoudi S., Antonchuk J., Zhao S, & Medvinsky A. (2016). Inductive interactions mediated by interplay of asymmetric signalling underlie development of adult haematopoietic stem cells. Nature Communications, 7, 10784.

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Comprehensive Antibody Panel for Molecular Analysis

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Primary antibodies against Shp2 (sc-280), Wt1 (sc-7385), PLZF (sc-28319), Cx43 (sc-9095), UPA (sc-14019), TPA (sc-81693), ABP (sc-32891), AR (sc-816), PDGFA (sc-128), DMRT1 (sc-10485), ERK1/2 (sc-292838), Akt1/2/3 (H-136, sc-8312) and AMH (sc-6885) were obtained from Santa Cruz Biotechnology, Dallas, Texas, USA. Antibodies for phospho-Akt (Ser473, 4060), phospho-ERK1/2 (Thr202/Thr204, 4370) and PH3 (9701s) were purchased from Cell Signaling Technology, Danvers, Massachusetts, USA. Antibodies against SCF (ab64677), Stra8 (ab49405), SCP3 (ab97672), Claudin11 (ab53041), FSHR (ab35309), GDNF (ab18956) and pem (ab31922) were purchased from Abcam, Cambridge, Massachusetts, USA. Other antibodies included JAMA (36–1700, Invitrogen, Grand Island, New York, USA) and BMP4 (SAB2700755, Sigma Chemical Co., St. Louis, Missouri, USA). All chemical reagents were purchased from Sigma-Aldrich and Solarbio (Shanghai, China).

Hu X., Tang Z., Li Y., Liu W., Zhang S., Wang B., Tian Y., Zhao Y., Ran H., Liu W., Feng G.S., Shuai J., Wang H, & Lu Z. (2015). Deletion of the tyrosine phosphatase Shp2 in Sertoli cells causes infertility in mice. Scientific Reports, 5, 12982.

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Characterizing Testicular Tissue Morphology

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Samples (1.5 cm³) of testicular tissue were collected and placed in Petri dishes with HBSS or DMEM/F-12 and then fixed with 4% paraformaldehyde (PFA) for 48 h at 4 °C, replacing the PFA once during the incubation. The tissue was then dehydrated and embedded, after which 4–6 μm-thick sections were cut and stained with hematoxylin and eosin for histological analysis. Additionally, the expression and localization of a number of marker genes were identified using immunohistochemical analysis using the following primary antibodies: rabbit anti-cyclin A1 (1:100 dilution, ab133183; Abcam, Cambridge, UK), rabbit anti-UCHL1 (1:50 dilution, bs-11677R; Boosen, Beijing, China), and rabbit anti-SCF (1:100 dilution, ab64677; Abcam, Cambridge, UK). Goat anti-rabbit IgG (1:50 dilution, SP-9001; Jin Qiao, Beijing, China) was used to catalyze the conversion of DAB to a brown precipitate during immunohistochemical staining. Testis morphology was then evaluated using 100× and 400× magnifications using a digital tricamera microscope.

Wu Y., Guo T., Li J., Niu C., Sun W., Zhu S., Zhao H., Qiao G., Han M., He X., Lu Z., Yuan C., Han J., Liu J., Yang B, & Yue Y. (2022). The Transcriptional Cell Atlas of Testis Development in Sheep at Pre-Sexual Maturity. Current Issues in Molecular Biology, 44(2), 483-497.

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Ovarian Tissue Protein Signaling Analysis

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Protein lysate from fresh ovarian tissue was prepared, separated on 10% sodium dodecyl sulfate–polyacrylamide gel, and transferred to a polyvinylidene difluoride membrane (Millipore, California, USA). The membranes were blocked with 5% nonfat milk in Tris–HCl (10mM, pH 7.4) containing 150mMNaCl and 1% Nonidet P-40 and separately incubated with the following specific antibodies at 4°C overnight: rabbit anti-Kitl polyclonal antibody (1:1000, ab64677; Abcam, Massachusetts, USA), rabbit anti-PTEN monoclonal antibody (1:1000, 9559; CST), rabbit anti-Aktmonoclonal antibody (1:1000, 4691; CST),rabbit anti-pAkt monoclonal antibody (Ser473, 1:1000, 4691; CST), rabbit anti-foxo3a monoclonal antibody(1:1000, 12829; CST), and rabbit anti-pfoxo3a polyclonal antibody(Ser253, 1:1000, 9466; CST). The relative intensity of protein bands was quantified by digital densitometry (ImageJ software, National Institutes of Health, Maryland, USA). The β-actin levels were used as internal standards.

Xu M., Sun J., Wang Q., Zhang Q., Wei C, & Lai D. (2018). Chronic restraint stress induces excessive activation of primordial follicles in mice ovaries. PLoS ONE, 13(3), e0194894.

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Mast Cell Recruitment in IVD

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To assess recruitment of mast cells into the IVD, IHC was performed for CCL2/MCP-1 (1:200 Abcam ab9669) and chemoattractant SCF (1:100 Abcam ab64677) on human autopsy and surgical tissue and imaged as described previously.

Wiet M.G., Piscioneri A., Khan S.N., Ballinger M.N., Hoyland J.A, & Purmessur D. (2017). Mast Cell-Intervertebral disc cell interactions regulate inflammation, catabolism and angiogenesis in Discogenic Back Pain. Scientific Reports, 7, 12492.

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Immunohistochemical Analysis of Skin and Hair

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Skin samples were fixed in 4% para-formaldehyde, washed in PBS, dehydrated and embedded in paraffin. Five-micrometer sections of hair, croup skin and dorsal midline skin were stained with hematoxylin and eosin. ImageJ was used to measure hair bulb area and hair color intensity. Color intensity was measured at six points across the diameter of the hair cortex and averaged for each hair. Hair color intensity measurements were taken from at least five hairs from each genotype. Immunofluorescence was carried out with goat antibody to TBX3 (Santa Cruz Biotechnology, sc-17871), mouse antibody to MITF (Abcam, ab12039), rabbit antibody to SCF (KITLG; Abcam, ab64677), mouse antibody to AE13 (Abcam, ab16113), rabbit antibody to Ki67 (Abcam, ab15580), mouse antibody to AE15 (Abcam, ab58755), rabbit antibody to KRT6 (Abcam, ab24646) and rabbit antibody to KIT (Dako, A4502) after antigen retrieval using Tris-EDTA, pH 9 (TBX3) or 0.01 M citrate buffer, pH 6 (MITF, KITLG, AE13, Ki67, AE15, KRT6 and KIT) in a pressure cooker. All photomicrographs are representative of at least two animals of each genotype and anatomical location.

Imsland F., McGowan K., Rubin C.J., Henegar C., Sundström E., Berglund J., Schwochow D., Gustafson U., Imsland P., Lindblad-Toh K., Lindgren G., Mikko S., Millon L., Wade C., Schubert M., Orlando L., Penedo M.C., Barsh G.S, & Andersson L. (2015). Regulatory mutations in TBX3 disrupt asymmetric hair pigmentation that underlies Dun camouflage color in horses. Nature genetics, 48(2), 152-158.

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Testis Tissue Protein Analysis

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The total proteins in the testis tissues were separated using a Total Protein Extraction Kit (BC3711, Solarbio). The protein concentration was measured using a Protein Assay kit (Beyotime). Next, the protein samples were separated by 10% SDS-PAGE and electrically transferred to PVDF membranes (Millipore, MA, USA). After sealing with 3% bovine serum albumin at room temperature for 1 h, the membranes were hatched with the primary antibodies (rabbit anti-prohibitin (PHB), ab28172, 1:1000; rabbit anti-Beclin-1, ab62557, 1:2000; rabbit anti-LC3II/I, ab128025, 1:1,000; rabbit anti-p62, ab56416, 1:1,000; rabbit anti-SCF, ab64677, 1:1000; rabbit anti-Parkin, ab15494, 1:1,000; rabbit anti-LKB1, ab199970, 1:1,000; rabbit anti-AMPKα, ab131512, 1:500; rabbit anti-phosphorylated AMPKα (p-AMPKα), ab133448, 1:10000; rabbit anti-ULK1, ab167139, 1:1000; rabbit anti-p-ULK1, ab203207, 1:1000; rabbit anti-β-actin, ab8227, 1:5000; Abcam, Cambridge, UK) overnight at 4 °C. The membranes were incubated with goat-anti-rabbit IgG (H + L)-HRP (1:10000, ab6721, Abcam) for 1 h at room temperature and then rinsed thrice with TBST thrice. Protein bands were visualized using an Electrochemilluminescence (ECL) chemiluminescence kit (WBULS0500; EMD Millipore), and band intensity was analyzed with Image-Pro Plus 6.0.

Li G., Xu Y., Li Y., Chang D., Zhang P., Ma Z., Chen D., You Y., Huang X, & Cai J. (2023). Qiangjing tablets ameliorate asthenozoospermia via mitochondrial ubiquitination and mitophagy mediated by LKB1/AMPK/ULK1 signaling. Pharmaceutical Biology, 61(1), 271-280.

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Quantifying Stem Cell Factor in Mouse Retinas

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Eye samples were prepared after mice with euthanized. Retinas were then isolated and homogenized in an ice-cold mixture of RIPA buffer (Beyotime, China) containing protease inhibitor cocktail (Beyotime). Extracts were separated using 12% sodium dodecyl sulfate poly-acrylamide gels and transferred onto polyvinylidene uoride membranes. Membranes were incubated in TBST (12.5 mM Tris-HCl, pH 7.6, 75 mM NaCl, 0.1% Tween 20) containing 5% fat-free milk for 1 hour at room temperature, then transferred into solution containing primary antibodies at 4°C overnight, and probed with indicated secondary antibodies in TBST for 2 hours at room temperature. Membranes were exposed on an Odyssey infrared imaging system with the Odyssey Application software V1.2.15 (LI-COR Biosciences, United States). All blots were analyzed by ImageJ (National Institutes of Health, United States). The relative levels of SCF were determined by normalizing against β-actin. The primary antibodies used were as follows: anti-SCF at 1:1000 (ab64677, Abcam), anti-β-actin at 1:1000 (ab179467, Abcam). The secondary antibody used was peroxidase-conjugated goat anti-rabbit IgG at 1:2000 (Beyotime).

Chen X., Li S., Liu X., Zhao J., Yang X., You R., & Wang Y. (2021). Stimulation of c-Kit+ Retinal Progenitor Cells by Stem Cell Factor Confers Protection Against Retinal Degeneration.

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