Ikon m

Proteins from each sample were separated on 1D SDS-PAGE 12.5% gels. The electrophoretic gels were then blotted, according to [54 (link)], onto nitrocellulose membranes (120 V, 1 h; Protran B85, GE Healthcare Life Science, Velizy-Villacoublay, France). The membranes were incubated 1 h in 2% milk in TBS-Tween (20 mM Tris-HCl pH 7.3, 150 mM NaCl, 0.2% Tween 20) and overnight at 4 °C in a mix of rabbit polyclonal antibodies directed against LbGAP (1:10,000), LbGAP2 (1:2000) or LbSPN (that recognizes both LbSPNm and LbSPNy) (1:2000) in 2% milk in TBS-Tween. The three polyclonal antibodies are described in [29 (link),43 (link),44 (link),55 (link)]. After three washes in TBS-Tween, the membranes were incubated 2 h with a secondary goat anti-rabbit IgG horseradish peroxidase conjugate (1:10,000; Sigma, St. Quentin Fallavier, France) in 2% milk in TBS-Tween, washed three times ins TBS-Tween and revealed with a luminescent substrate (LuminataTM Crescendo Western HRP Substrate; Millipore, Burlington, MA, USA). Digital images of the blots were then acquired with a cooled CCD camera (Andor iKon-M, Abington-on-Thames, UK).

After floating-slice HCR, slices were mounted between no.1 coverslips with antifade compound (ProLong Glass, Invitrogen) and images were collected on an Andor CSU-X spinning disk confocal system coupled to a Nikon Eclipse Ti microscope equipped with an Andor iKon-M camera. The images were acquired with an oil immersion objective at 60×. The Alexa 594 patched cell backfill channel (561 nm) plus associated HCR probe/hairpin channels (488 nm and 647 nm) were projected through a 10–20-μm thick z-series so that an unambiguous determination of the association between the patch-filled cell and its HCR gene expression could be made. Images were processed using Nikon NIS Elements 4.4 and Nikon NIS AR.