Immunofluorescence Profiling of RNA-Binding Proteins

For immunofluorescence, cells were fixed in 10% neutral formalin and incubated overnight with the primary antibodies^{68 (link)}: anti-Sfpq, anti-Pspc1, or anti-Ago2 antibodies. Slides were coverslipped in Vectashield Mounting Medium with Dapi (Invitrogen). As control for immunofluorescence, slides were incubated with only the Alexa Fluor 488-conjugated anti-mouse or Alexa Fluor 594-conjugated anti-rabbit antibodies but not with primary antibodies. No signal was detected in these conditions. The results were analyzed on a Leica DM5500B microscope with a HAMAMATSU camera ORGA-ER.

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Bottini S., Hamouda-Tekaya N., Mategot R., Zaragosi L.E., Audebert S., Pisano S., Grandjean V., Mauduit C., Benahmed M., Barbry P., Repetto E, & Trabucchi M. (2017). Post-transcriptional gene silencing mediated by microRNAs is controlled by nucleoplasmic Sfpq. Nature Communications, 8, 1189.

Publication 2017

Dapi DM5500B microscope

Ago2 Alexa 594 Alexa fluor 488 Anti antibodies Antibodies Cells Dapi Formalin Immunofluorescence Microscope Mouse Rabbit

Corresponding Organization :

Other organizations : Inserm, Institut de Pharmacologie Moléculaire et Cellulaire, Université Côte d'Azur, Centre National de la Recherche Scientifique, Aix-Marseille Université, Institut de Recherche sur le Cancer et le Vieillissement de Nice

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Variable analysis

independent variables

Primary antibody used: anti-Sfpq, anti-Pspc1, or anti-Ago2 antibodies

dependent variables

Immunofluorescence signal detected

control variables

Cell fixation in 10% neutral formalin
Incubation duration for primary antibodies (overnight)
Mounting medium used (Vectashield Mounting Medium with Dapi)
Microscope and camera used (Leica DM5500B microscope with HAMAMATSU camera ORGA-ER)

controls

Positive control: Slides incubated with Alexa Fluor 488-conjugated anti-mouse or Alexa Fluor 594-conjugated anti-rabbit antibodies (no signal detected)
Negative control: Slides incubated with only the Alexa Fluor 488-conjugated anti-mouse or Alexa Fluor 594-conjugated anti-rabbit antibodies but not with primary antibodies (no signal detected)

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