PLA was performed according to the manufacturer’s protocol (Sigma,
DUO92102)46 (link). Briefly, ALI day 4 cultures from
E2f4f/f;
R26CreERT2/+ with or without Tamoxifen
treatment were fixed with 4% PFA room temperature for 10 min.
Samples were incubated with primary antibodies (anti E2f4, anti Deup1 or the
relevant control IgGs, as indicated) using the immunofluorescence microscopy
protocol described above, followed by incubation with the PLA probe set
(anti-rabbit plus strand/anti-mouse minus strand) at 37 °C for
2 h. Ligation of probes in close proximity was carried out at
37 °C for 30 min, followed by a 100-min amplification step.
Subsequently, we performed immunofluorescence staining for anti-Pcm1 antibody as
described above. All processed slides were mounted using ProLong Gold antifade
reagent (Life Technologies, catalogue no. P36930), and images were captured
using a confocal microscope system (Carl Zeiss, LSM 710).
DUO92102)46 (link). Briefly, ALI day 4 cultures from
E2f4f/f;
R26CreERT2/+ with or without Tamoxifen
treatment were fixed with 4% PFA room temperature for 10 min.
Samples were incubated with primary antibodies (anti E2f4, anti Deup1 or the
relevant control IgGs, as indicated) using the immunofluorescence microscopy
protocol described above, followed by incubation with the PLA probe set
(anti-rabbit plus strand/anti-mouse minus strand) at 37 °C for
2 h. Ligation of probes in close proximity was carried out at
37 °C for 30 min, followed by a 100-min amplification step.
Subsequently, we performed immunofluorescence staining for anti-Pcm1 antibody as
described above. All processed slides were mounted using ProLong Gold antifade
reagent (Life Technologies, catalogue no. P36930), and images were captured
using a confocal microscope system (Carl Zeiss, LSM 710).
Free full text: Click here
