After blocking and permeabilization, cryosections were incubated with primary antibodies against VEGFR-2 (rabbit, polyclonal, 1:100, ab39256; Abcam) and placed in a fridge (4°C) overnight. After washing with PBS, secondary FITC-coupled anti-rabbit IgG (goat, 1:400, F6005; Sigma Aldrich) or Alexa Fluor 488-coupled anti-rabbit IgG (goat, 1:400, A-11008, Molecular Probes) antibodies were added overnight at 4°C. Nuclear counterstaining was done with bisBenzimide Hoechst 33342 trihydrochloride (1:1000, B2261; Sigma-Aldrich). Further steps of immunohistochemistry were done in the same manner as just described (group 1 + 2) to label PCs.
F6005
Manufactured by Merck Group
Sourced in United States, Germany
The F6005 is a laboratory centrifuge designed for general-purpose applications. It is capable of processing a variety of sample types and volumes. The centrifuge can achieve a maximum speed of 6,000 rpm and has a maximum relative centrifugal force (RCF) of 4,000 x g. The device is equipped with a rotor that can accommodate 6 to 8 samples simultaneously. The F6005 is suitable for a range of laboratory procedures that require sample separation and concentration.
Automatically generated - may contain errors
Lab products found in correlation
G9023, by Merck Group (2 mentions)
P1951, by Merck Group (2 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
Ab39256, by Abcam (1 mentions)
Bisbenzimide hoechst 33342 trihydrochloride, by Merck Group (1 mentions)
B2261, by Merck Group (1 mentions)
Fluoromount g mounting media, by Thermo Fisher Scientific (1 mentions)
Eclipse 90i, by Nikon (1 mentions)
Nis element, by Nikon (1 mentions)
Ab11826, by Abcam (1 mentions)
Ab75855, by Abcam (1 mentions)
F1804, by Abcam (1 mentions)
T5393, by Merck Group (1 mentions)
Bisbenzimide h 33342 trihydrochloride, by Merck Group (1 mentions)
Extravidin fitc, by Merck Group (1 mentions)
S302380 2, by Agilent Technologies (1 mentions)
Goat anti rabbit igg, by Merck Group (1 mentions)
4 protocols using f6005
1
Immunohistochemical Localization of VEGFR-2
2
Immunofluorescence Assay for HeLa Cells
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HeLa cells were seeded on coverslips and transfected with 0.25 μg of plasmid DNA. Cells were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in PBST (PBS + 0.01% Tween 20) for 10 min at room temperature. The cells on coverslips were blocked with blocking solution (2% BSA/PBST) for 30 min and incubated with the primary antibodies anti-Flag (mouse, Sigma, M2, F1804), anti-PABPN1 (rabbit, Abcam, ab75855), anti- SC35 (mouse, Abcam, ab11826) diluted in the blocking solution overnight at 4 °C. Proteins were visualized with the FITC- and TRITC-conjugated secondary antibodies (Sigma, F6005, and T5393) for 1 h at room temperature. Nuclei were stained with DAPI-supplemented Fluoromount-G mounting media (Thermo Fisher Scientific). Cells were visualized with a fluorescence microscope (Nikon eclipse 90i), and the images were analyzed with the NIS-elements (Nikon) software.
3
Visualizing Cx43 in Glioblastoma Cytoskeleton
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To confirm the presence of Cx43 in U-87 and U-251 actin cytoskeleton specifically, GBM cells were fixed with 4% PFA for 30 minutes followed by permeabilization with 0.3% Triton for 10 minutes. The unspecific binding sites were blocked with goat-serum (Sigma-Aldrich, G9023, 1:50 in PBS, 30 minutes). After washing, cells were incubated with anti-connexin 43 (Merck Millipore, MAB3068, USA, 1:100 in PBS) overnight, followed by incubation with FITC-coupled anti-rabbit IgG (Sigma-Aldrich, F6005, 1:200 in PBS, 1.5 hours) and subsequently treated with rhodamin-phalloidin (Sigma-Aldrich, P1951, 1:20 in PBS, 1 hour). Then bisBenzimide H 33342 trihydrochloride (Hoechst, Sigma-Aldrich, B2261, 1:1,000 in PBS, 20 minutes) was applied to counterstain the cell nuclei.
To visualize cell coupling, microinjected cells were fixed with 4% PFA plus 0.2% picrinic acid for 2 hours. After several washing steps, cells were incubated with ExtrAvidin FITC (Sigma-Aldrich, E2761, 1:100 in PBS, 2 hours) to visualize neurobiotin. To count the cells, bisBenzimide H 33342 trihydrochloride (Hoechst, Sigma-Aldrich, B2261, 1:1,000 in PBS, 20 min) was applied to stain the nucleus. Finally the cover slips were mounted on microscope slides with fluoromount mounting medium.
To visualize cell coupling, microinjected cells were fixed with 4% PFA plus 0.2% picrinic acid for 2 hours. After several washing steps, cells were incubated with ExtrAvidin FITC (Sigma-Aldrich, E2761, 1:100 in PBS, 2 hours) to visualize neurobiotin. To count the cells, bisBenzimide H 33342 trihydrochloride (Hoechst, Sigma-Aldrich, B2261, 1:1,000 in PBS, 20 min) was applied to stain the nucleus. Finally the cover slips were mounted on microscope slides with fluoromount mounting medium.
4
Immunofluorescent Labeling of DRG Explants
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DRG explants were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.015% Triton-X-100 in phosphate-buffered saline (PBS) for 5 min. After three washing steps with PBS, non-specific binding sites were blocked by incubation for 30 min with 2% (w/v) goat serum in PBS (Sigma-Aldrich, G9023, Darmstadt, Germany). Explants were again washed in PBS three times, and were incubated either with anti-cofilin [34 (link)] (1:100 in PBS) or anti-Arp2 (1:100 in PBS, Cell signaling technology, 3128S, Frankfurt am Main, Germany) overnight at 4 °C. The samples were then incubated with the secondary FITC-conjugated antibody (goat anti-rabbit IgG, Sigma-Aldrich; F6005, Darmstadt, Germany) diluted to 1:200 in PBS for two hours at room temperature. To label actin, cells were finally treated with phalloidin rhodamine (1:20 in PBS, Sigma-Aldrich, P1951, Darmstadt, Germany) for 30 min. Finally, cell cultures were rinsed in PBS and coverslipped in mounting medium (Dako, S302380-2, Agilent, Santa Clara, CA, USA).
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