Passage 3 (P3) KSCs were fixed in 4% paraformaldehyde/PBS buffer in a 24-well plate (Corning Inc., USA) at room temperature, permeabilized with 0.1% Triton X-100 (Beijing Chemical Factory), blocked for 30 min in 10% goat serum (BD, USA), and then incubated with primary antibodies at 4°C overnight. The primary antibodies included mouse anti-E-cadherin polyclonal antibody, rabbit anti-ZO-1 polyclonal antibody, rabbit anti-fibronectin polyclonal antibody (Santa Cruz, USA), mouse anti-N-cadherin polyclonal antibody, mouse anti-α-SMA polyclonal antibody (Sigma, USA), and rabbit anti-Vimentin polyclonal antibody (Cell Signaling Technology, USA). The secondary antibodies—tetramethylrhodamine isothiocyanate (TRITC)-labeled goat anti-rabbit IgG, goat anti-mouse IgG, fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG, or goat anti-mouse IgG (Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd., China)—and 4′-6-diamidino-2-phenylindole (DAPI, BD, USA) were added sequentially. Imaging was performed with an IX70 confocal fluorescence microscope (Olympus, Japan).
Goat anti mouse igg
Manufactured by Zhongshan Golden Bridge Biotechnology
Sourced in China
Goat anti-mouse IgG is a secondary antibody produced by immunizing goats with mouse immunoglobulin G (IgG). This antibody is designed to specifically recognize and bind to mouse IgG, allowing it to be used as a detection tool in various immunoassays and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg, by Zhongshan Golden Bridge Biotechnology (4 mentions)
Rapamycin, by Merck Group (1 mentions)
Rabbit anti mtor antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti p mtor ser2448, by Cell Signaling Technology (1 mentions)
Mouse anti e cadherin antibody, by Santa Cruz Biotechnology (1 mentions)
Mouse anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti p akt ser473, by Cell Signaling Technology (1 mentions)
Rabbit anti akt antibody, by Cell Signaling Technology (1 mentions)
Ly294002, by Merck Group (1 mentions)
Tris acetate gel, by Thermo Fisher Scientific (1 mentions)
Mouse anti actin, by Abcam (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Hrp conjugated secondary antibody, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Goat anti rat igg, by Zhongshan Golden Bridge Biotechnology (1 mentions)
Goat serum, by BD (1 mentions)
Rabbit anti vimentin polyclonal antibody, by Cell Signaling Technology (1 mentions)
Mouse anti α sma polyclonal antibody, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole dapi, by BD (1 mentions)
24 well plate, by Corning (1 mentions)
7 protocols using goat anti mouse igg
1
Immunofluorescence Staining of KSCs
2
Adenoviral Delivery of S100A4 and PI3K/Akt Signaling
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Adenovirus expressing S100A4 and green fluorescent protein (Ad-S100A4), and adenovirus expressing green fluorescent protein (Ad-GFP) were kindly provided by Dr. Tongchuan He (Medical Center, Chicago University, Chicago, USA). The PI3K/Akt inhibitor LY294002 and the mTOR/p70S6K inhibitor rapamycin were purchased from Sigma-Aldrich (Saint Louis, MO, USA). All antibodies used were: goat anti-S100A4 antibody (Cat#19948, Santa Cruz, CA, USA), mouse anti-β-actin antibody (Cat#47778, Santa Cruz, CA, USA), mouse anti-E-cadherin antibody (Cat#8426, Santa Cruz, CA, USA), rabbit anti-Akt antibody (Cat#4691, Cell Signaling, MA, USA), rabbit anti-p-Akt (Ser473) (Cat#4060, Cell Signaling, MA, USA), rabbit anti-mTOR antibody (Cat#2983, Cell Signaling, MA, USA), rabbit anti-p-mTOR (Ser2448)(Cat#2971, Cell Signaling, MA, USA), rabbit anti-p70S6K antibody (Cat#2708, Cell Signaling, MA, USA), rabbit anti-p-p70S6K (Thr421/Ser424)(Cat#9204, Cell Signaling, MA, USA), rabbit anti-goat IgG (Cat#2306, Zhongshan Golden Bridge, Beijing, China), goat anti-mouse IgG (Cat#2305, Zhongshan Golden Bridge, Beijing, China), goat anti-rabbit IgG (Cat#2301, Zhongshan Golden Bridge, Beijing, China).
3
Circadian Protein Rhythms in Drosophila
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Flies were synchronized for 3 days under LD (500 lux:0 lux) cycles, and collected every 3–4 h. Protein was extracted from 40–50 male heads at each time point. Frozen heads were homogenized in 150 ml ice-cold extraction buffer (20 mM HEPES, pH 7.5, 100 mM KCl, 5% glycerol, 10 mM EDTA, 0.1% Triton, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) plus protease inhibitors (complete mini Roche), phosphatase inhibitors cocktails 1 (0.5%) and 2-mM b-glycerophosphate (1%; Sigma). For SDS–PAGE, 50 ug of protein extracts were separated on 8–10% Tris–acetate gels (Invitrogen) and transferred to NC membranes. Primary antibodies used were mouse anti-ACTIN (abcam;1∶2000) and rat ani-CRY and rat anti-TIM (from the laboratory of Dr. Rosbash Lab; anti-CRY 1∶500; anti-TIM 1∶1000). HRP-conjugated secondary antibodies (Zhongshan Goldenbridge Biotechnology Co., Ltd) were goat anti-mouse IgG (1∶1000) and goat anti rat IgG (1∶1000). All western blots were performed in triplicate. Band intensity was calculated and analyzed with the Gel-Pro Analyzer 4.0.
4
Immunohistochemical Analysis of DDX56 and Ki-67
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Immunohistochemical specimens were fixed in 10% formaldehyde, which were then embedded in wax blocks. This study obtained anti-DDX56 antibody in Santa Cruz Biotechnology (5A7: sc-101,018, Santa Cruz, CA) and Ki-67 polyclonal antibody in Proteintech Group (No. 26,593-1-AP, ProteinTech, Wuhan, China). Following manufacturer’s instruction, the endogenous peroxidase blocking reagent (SP-9000, Zhongshan Golden Bridge, Beijing, China), goat serum (SP-9000, Zhongshan Golden Bridge), anti‐DDX56 antibody, Ki-67 polyclonal antibody, goat anti-mouse IgG (SP-9000, Zhongshan Golden Bridge), goat anti-rabbit IgG (SP-9000, Zhongshan Golden Bridge), HRP-labeled avidin working fluid (SP-9000, Zhongshan Golden Bridge) and Diaminobenzidine (DAB; ZLI-9018, Zhongshan Golden Bridge) were added in a regular sequence. Finally, the tissue sections were stained, and then a light microscope (Olympus Corporation, Tokyo, Japan) was employed for observation. Tumor histology was independently reviewed by an experienced pathologist. The DDX56 levels were rated as 0–3, indicating negative, low, moderate, and high intensities, respectively. The scores regarding the extent of staining were 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). In addition, a score of >4 was suggested as high.
5
Antibodies and Apoptosis Assay for Cancer
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TBX2 polyclonal antibodies were purchased from Bioworld Technology, Inc., St. Louis Park, MN, USA. Polyclonal mouse antibodies against E-cadherin, N-cadherin, Vimentin and Fibronectin were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Snail and Twist polyclonal rabbit antibodies were purchased from ABclonal Biotechnology Co., Ltd., Cambridge, MA, US. Mouse monoclonal antibody against Mdm2, p53, Bax, Akt, p-Akt, VEGF and polyclonal rabbit antibodies against Bcl-2 and β-actin were all purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA. The secondary antibodies including goat anti-rabbit IgG and goat anti-mouse IgG were obtained from Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China. The standard of dilution was referenced in accordance to the antibody specification. TBX2 siRNA and negative control siRNA were designed, synthesized by GenePharma (Suzhou, China). Cell proliferation was measured by Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology, Nantong, China). Cell migration and Cell invasion ability were tested using transwell chambers (Corning Incorporated, Corning, NY, USA). Cell apoptosis was assessed by Annexin V-FITC/propidium iodide (PI) Apoptosis Detection kit (KeyGen, Nanjing, China).
6
Immunohistochemical Analysis of HMGB1 Expression
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After the electrophysiology studies, the brain was rapidly removed, and samples were immersed in 10% formalin for 24 h, embedded in paraffin and cut into 5-μm thick slices. The paraffin sections were deparaffinized, rehydrated and soaked in 0.1 M of citric acid buffer for 15 min at 92–98°C in a microwave oven, and washed with PBS. Then, the sections were incubated with the primary antibodies of anti-HMGB1 (ab172730-Rabbit monoclonal IgG, 1:5,000; Abcam) overnight at 4°C. Subsequently, the samples were incubated with horseradish peroxidase-conjugated secondary antibodies of rabbit anti-sheep IgG (KPL, Gaithersburg, MD, USA) and goat anti-mouse IgG (Zhongshan Golden Bridge Biotechnology) for 1 h at 37°C. Immunore-activity was developed with 3,3′-diaminobenzidine tetrahydrochloride (Zhongshan Golden Bridge Biotechnology). Finally, the sections were counter-stained with hematoxylin, mounted and examined by microscopy.
7
Quantitative Western Blot Analysis
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Total protein was extracted from each of the six groups' cells with radioimmunoprecipitation assay (RIPA). Following centrifugation, the protein concentration was determined by bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, Jiangsu, China). Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions and transferred to 0.22-µm polyvinylidene difluoride (PVDF) membranes (Millipore Inc., Billerica, MA, USA). Membranes were blocked in 5% milk powder for 1 h. After washing the membranes thrice with tris-buffered saline with Tween 20 (TBST), antibodies FZD4, FZD5, FZD7, c-myc, and GAPDH (Sigma, St. Louis, MO, USA), they were incubated overnight. Goat anti-rabbit IgG (Zhongshan Golden Bridge Biotechnology, Beijing, China) and Goat anti-mouse IgG (Zhongshan Golden Bridge Biotechnology) were used as secondary antibodies. Target proteins were detected with the Immobilon Western (Millipore). Each experiment was conducted in triplicate and repeated three times. Densitometric analyses were performed using ImageJ software (National Institutes of Health (NIH)).
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