Routine Cellular Characterization Techniques

Routine tissue culture, FACS analysis, immunofluorescence and confocal microscopy were performed as described previously (Faunes et al., 2013 (link); Kalmar et al., 2009 (link); Turner et al., 2014a (link),b (link),c (link)). Primary antibodies used for immunofluorescence were: goat anti-Bra (Santa Cruz Biotechnology, sc-17743; 1:200), rat anti-E-Cadherin (Takara, M108; 1:200), goat anti-Sox17 (R&D Systems, AF1924; 1:500) and goat anti-FoxA2 (Santa Cruz Biotechnology, sc-6554; 1:500). Alexa-conjugated secondary antibodies were from Invitrogen and were used at 1:500 dilution. Hoechst 3342 (Invitrogen) stained the nuclei and was used at 1:1000 dilution.

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van den Brink S.C., Baillie-Johnson P., Balayo T., Hadjantonakis A.K., Nowotschin S., Turner D.A, & Martinez Arias A. (2014). Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells. Development (Cambridge, England), 141(22), 4231-4242.

Publication 2014

goat anti-Bra rat anti-E-Cadherin goat anti-Sox17 goat anti-FoxA2 Alexa-conjugated secondary antibodies Hoechst 3342

Antibodies Confocal microscopy Dilution Goat Immunofluorescence Nuclei Rat e cadherin Tissue

Corresponding Organization :

Other organizations : University of Cambridge

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Protocol cited in 4 other protocols

Variable analysis

independent variables

Routine tissue culture
FACS analysis
Immunofluorescence
Confocal microscopy

dependent variables

Protein expression levels (measured by immunofluorescence and confocal microscopy)

control variables

Experimental procedures as described in the referenced publications (Faunes et al., 2013; Kalmar et al., 2009; Turner et al., 2014a, b, c)
Primary antibodies used for immunofluorescence (goat anti-Bra, rat anti-E-Cadherin, goat anti-Sox17, goat anti-FoxA2)
Alexa-conjugated secondary antibodies used at 1:500 dilution
Hoechst 3342 (Invitrogen) used to stain nuclei at 1:1000 dilution

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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