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Membrane androgen receptor activation triggers pro-apoptotic responses in vitro and in vivo and blocks migration in colon cancer

von S. G.

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[1.] Shg/Fragment 043 02 - Diskussion
Zuletzt bearbeitet: 2014-11-03 19:55:26 Graf Isolan
BauernOpfer, Fragment, Gesichtet, Gu et al 2009, SMWFragment, Schutzlevel sysop, Shg

Typus
BauernOpfer
Bearbeiter
SleepyHollow02
Gesichtet
Yes
Untersuchte Arbeit:
Seite: 43, Zeilen: 2-3, 11-20
Quelle: Gu et al 2009
Seite(n): 3, Zeilen: li. Sp. 11-12, (13-27), 27ff
3.2.4. Immunofluorescence analysis and confocal laser scanning microscopy

For testosterone-HSA-FITC staining, 5-μm-thick frozen tissue sections from the Balb/c or APC mouse tumors were fixed with 4% PFA for 15 min and incubated with 5% BSA/1x PBS/0.3% Triton for 1 hour at room temperature. After two washes with PBS 1.5% FBS specimens were exposed to testosterone-HSA-FITC (10-7 M, Sigma) for 1h at room temperature. Nuclei were stained with DRAQ-5 dye (1:1000, Biostatus, Leicestershire, UK) for 10 min at room temperature.

For direct fluorescence microscopy of F-actin, cells were fixed with 3 % paraformaldehyde in PBS for 30 min, permeabilized with 0.5 % Triton X-100 in PBS (10 min) and incubated with rhodamine-phalloidin (Molecular Probes, Eugene, OR, 1:100 dilution) for 40 min in the dark. For indirect immunofluorescence staining, cells were incubated for 2h at room temperature with mouse monoclonal anti-tubulin (Cell signaling, 1: 1000 dilution). Secondary FITC-conjugated rabbit anti-mouse IgG (Invitrogen) was used in a 1: 200 dilution. Nuclei were stained with DRAQ5™ (Biostatus Limited). Slides were mounted using the ProLang® Gold Antifade reagent (Invitrogen).

Immunofluorescence analysis and confocal laser scanning microscopy

Cells were cultured on glass cover slips with testosterone- HSA-FITC or control HSA-FITC using the concentrations and the incubation periods indicated in the figure legends. For testosterone-HSA-FITC staining, cells or specimens were washed twice with PBS containing 1.5% FBS for 1.5 min and incubated for 1 h with 1% BSA in PBS at room temperature. After two washes with PBS/1.5% FBS cells were exposed to 10-7 M testosterone-HSA-FITC, while control cells were incubated with 4 × 10-7 M HSA-FITC for 1 h at room temperature. Nuclei were stained with DRAQ5™ (Biostatus Limited) or TO-PRO-3 (Invitrogen). After two washes with PBS/1.5% FBS and fixation with 0.5% paraformaldehyde for 30 min cells were washed twice with PBS/1.5% FBS for 3 min and mounted with slow anti-fade. For direct fluorescence microscopy of F-actin, cells were fixed with 3% paraformaldehyde in PBS for 30 min, permeabilized with 0.5% Triton X-100 in PBS (10 min) and incubated with rhodamine-phalloidin (Molecular Probes, Eugene, OR, 1:100 dilution) for 40 min in the dark. For indirect immunofluorescence staining cells were incubated for 2 h at room temperature with mouse monoclonal anti-tubulin (Cell signaling, 1: 1000 dilution). Secondary FITC-conjugated rabbit anti-mouse IgG (Invitrogen) was used at a 1:200 dilution. Nuclei were stained with DRAQ5™ (Biostatus Limited). Slides were mounted using the ProLang® Gold Antifade reagent (Invitrogen).

Anmerkungen

Obwohl Shg als Coautor von Gu et al (2009) genannt wird, stammt keine der Formulierungen dieses Artikels von Shg (vgl. die Anmerkungen zu Quelle:Shg/Gu_et_al_2009).

Ergo: Insbesondere im zweiten Absatz liegt die Übernahme eines Fremdtextes ohne jede Kennzeichnung vor.

Ferner: aus dem "10-7" der Vorlage wird bei Shg "10-7".

Gu et al 2009 has been mentioned once on page 41 with respect only to "previous titration experiments".

Sichter
(SleepyHollow02), Graf Isolan


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