Immunoblotting was performed as previously described [44 (link)]. Equal amounts of tissue lysates were used for immunoblotting. Blots were incubated with specific antibodies to BMP-1 and LTBP-1 (Abcam, ab205394 and ab78294), Flag (Sigma-Aldrich, F3165), ALK5 (R&D system, MAB5871), Col3a1 and Fibronectin 1 (Novus Biologicals, NB600 and NBP1–91258). β-Actin (Sigma-Aldrich, A2228) was used as a loading control. Immunofluorescence was performed as previously described in detail [44 (link)]. We used specific antibodies to CD34 (BD Bioscience, 553731 or Abcam, ab54208), Nkx2.1 (Abcam, ab76013) and VE-cadherin (BD biosciences, 562243). The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, D9564).
Ab54208
Manufactured by Abcam
Ab54208 is a laboratory instrument designed for specific applications. It measures 10 x 10 x 15 cm and weighs 2 kg. The product details and technical specifications are available from the manufacturer.
Automatically generated - may contain errors
Lab products found in correlation
D9564, by Merck Group (2 mentions)
A2228, by Merck Group (2 mentions)
Nbp1 91258, by Novus Biologicals (2 mentions)
β actin, by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Ve cadherin, by BD (1 mentions)
Ab119339, by Abcam (1 mentions)
Hoechst 33258, by Serva Electrophoresis (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (1 mentions)
Ab92547, by Abcam (1 mentions)
Ab76013, by Abcam (1 mentions)
Ab205394, by Abcam (1 mentions)
Ab78294, by Abcam (1 mentions)
Mab5871, by R&D Systems (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
F3165, by Merck Group (1 mentions)
3 protocols using ab54208
1
Immunoblotting and Immunofluorescence of BMP-1, LTBP-1, and Markers
2
Immunofluorescence Staining of Spheroid Cultures
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Spheroids in fibrin gel were fixed in 4% paraformaldehyde (+4°C, overnight), washed thrice in PBS, permeabilized by 0.2% Triton X-100 (in PBS) for 10 min, and blocked with 5% goat serum in PBS. Samples were then incubated (+4°C, overnight) in the mix of PBS+0.1% Tween-20+5% goat serum with primary antibodies against CD31 (ab119339; Abcam), fibronectin (MA5-11981; Abcam), CD34 (ab54208; Abcam), and vimentin (ab92547; Abcam). After three times washing with PBST (PBS+0,1% tween), samples were then incubated in 250 μl of a solution of secondary species-specific antibodies conjugated with fluorochromes Alexa Fluor 488 and Alexa Fluor 594 (ThermoScientific, United States). All antibodies were used at 1/500 dilution. The nuclei were stained with 2 μg/ml of intercalating dye Hoechst 33258 (Serva, Germany) or DAPI (Abcam). The preparations in the mounting medium were covered with coverslips and examined under LSM 880 laser confocal scanning microscope (ZEISS, Germany) in visible and UV light.
3
Immunoblotting and Immunofluorescence Analysis
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Immunoblotting was performed as described previously [44 (link)]. Equal amounts of tissue lysates were used for immunoblotting. Blots were incubated with specific antibodies to BMP-1 and latent TGFβ1-binding protein-1 (LTBP-1) (ab205394 and ab78294; Abcam), Flag (F3165; Sigma-Aldrich), ALK5 (MAB5871; R&D Systems), Col3a1 and Fn1 (NB600 and NBP1-91258; Novus Biologicals). β-Actin (A2228; Sigma-Aldrich) was used as a loading control. Immunofluorescence was performed as described in detail previously [44 (link)]. We used specific antibodies to CD34 (553731; BD Bioscience or ab54208; Abcam), Nkx2.1 (ab76013; Abcam) and VE-cadherin (562243; BD Biosciences). The nuclei were stained with 4′,6-diamidino-2-phenylindole (D9564; Sigma-Aldrich).
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