Zen 2.1 blue edition software

The light microscopic observations of histological or immunohistochemical staining were made by use of a BX50 microscopy (Olympus, Tokyo, Japan) equipped with an Axiocam ERc 5 s camera (Carl Zeiss, Obercohen, Germany) and an SE64 Rel.4.9.1. SP2 software (Carl Zeiss). The objective lenses used were UPlanApo 10x/0.40 and UPlanApo 20x/0.70 (Olympus). The observations of immunofluorescent staining were made by use of a confocal laser-scanning microscope (LSM800, Carl Zeiss) equipped with a Zen 2.1 [blue edition] software (Carl Zeiss). The objective lens used was Plan-Apochromat 20x/0.8 M27.

HEK293T cells grown on polyethylene coverslips (Thermo Fisher Scientific, Vilnius, Lithuania) were co-transfected with the expression plasmids for Staufen-2 FLAG or c-Flag pcDNA3 with A3G HA or Staufen-1 HA or HIV-1 Gag EGFP using Lipofectamine LTX and Plus reagent (Invitrogen, Karlsruhe, Germany). After 24 h of transfection, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min, permeabilized with 0.1% Triton X-100 for 10 min and incubated with blocking solution (10% FBS in PBS) for 1 h. Then, cells were stained with rabbit anti-Flag antibody (Sigma-Aldrich, Taufkirchen, Germany) in a 1:1000 dilution in blocking solution for 1 h and mouse anti-HA (Sigma-Aldrich, Taufkirchen, Germany) antibody in a 1:1000 dilution in blocking solution for 1 h. Secondary antibodies, Donkey anti-rabbit Alexa Fluor 594 (Thermo Fisher Scientific, Baltics, UAB) followed by Donkey anti-mouse Alexa Fluor 488 (Covance, Münster, Germany), were used at a 1:300 dilution in blocking solution for 1 h. Finally, DAPI was used to stain nuclei for 2 min. The images were captured using a 60× objective on a Zeiss LSM 510 Meta laser scanning confocal microscope (Carl Zeiss, Cologne, Germany). The images were analyzed by ZEN 2.1 (blue edition) software (Carl Zeiss).

Sections of 20 μm thickness were stained in immunofluorescence with goat anti-human cathepsin B (AF965; 10 μg/ml, R&D Systems) or anti-mouse FABP4 (AF1443; 10 μg/ml, R&D Systems) and Cy3-conjugated donkey anti-goat IgG (AP180C; 1:200, Merck Millipore), or rabbit anti-rat FABP5 (0.5 μg/ml, Owada et al. 2001 (link)) and Cy3-conjugated donkey anti-rabbit IgG (AP182C; 1:200; Merck Millipore). Sections were subsequently treated with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI, ImmunoBioScience) for nuclear staining. 3D images and movies were reconstructed by use of Zen 2.1 [blue edition] software (Carl Zeiss).