SIPA1L1/SPAR1 Interacts with the Neurabin Family of Proteins and is Involved in GPCR Signaling. Matsuura et al. Raw data for super-resolution colocalization analyses.
Super-resolution microscopic images stained for SIPA1L1 and its candidate interacting-proteins in the neuropil region of layer V cerebral cortex or hippocampal CA1 area were acquired using Olympus SD-OSR, a confocal-based spinning disk super-resolution microscope, which implements structured illumination microscopy (SIM). Four serial Z-stack images (200-nm step size) of 1024 x 1024 pixels (41 x 41 um2)/image were used for colocalization analyses. w1 files are for SIPA1L1 and w2 files for others in all folders with the exception of drebrin and actinin where SIPA1L1 is w2.
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Mice were deeply anesthetized with 90 mg/kg sodium pentobarbital and were intracardially perfused with ice-cold sodium phosphate buffer (pH 7.3, NPB), followed by ice-cold 4% paraformaldehyde (PFA) /NPB. The whole brain was removed, separated bilaterally at the medial line and fixed in ice-cold 4% PFA/NPB for 2 h. The brain was further infiltrated sequentially with 10, 15, and 20% sucrose/NPB for more than 4 h at each concentration and then frozen in a Tissue-Tek OCT compound (Sakura Finetek). 10 um cryosections were attached to an MAS-coated slide glass (S9441 Matsunami) and air dried for 2 h. For permeabilization, sections were incubated in 0.3% Triton X-100/Tris-buffered saline (pH7.5, TBS) for 10 min at room temperature (RT) except for synaptophysin and spinophilin staining, which sections were incubated in boiling 10 mM sodium citrate for 5 min. Sections were blocked with TBS containing a 0.5% blocking reagent (Roche), 2% fetal bovine serum, and 0.1% Tween-20 for 1 h. Then they were incubated overnight at 4°C with primary antibodies diluted in the blocking buffer. In the case of reactions containing mouse antibodies, reagents from the VECTOR M.O.M. Basic Kit (Vector Laboratories) were added. Following washes in TBS containing 0.1% Tween-20 (TBST), sections were incubated for 1 h at RT with secondary antibodies diluted in blocking buffer. Sections were subsequently washed, and coverslipped with Vectashield mounting medium (Vector Laboratories). Digital images were obtained using SD-OSR (Olympus) equipped with Yokogawa CSU-W1 scanner, Hamamatsu Orca Flash 4 V2+ High Speed SCMOS camera, and x 100, 1.35 NA silicone immersion objective with correction collar (UPLSAPO100XS) was used to acquire Z-stack images (200-nm step size, all channels scanned in each plane) of 1024 x 1024 pixels (41 x 41 um2)/image.