Nis elements ar imaging software

For the imaging of the GFP fusion proteins, Nile red, DASPMI and DAPI staining a 100x Plan Apochromat objective (NA = 1.4) by Nikon (Tokyo, Japan) connected to an Eclipse Ni-U microscope equipped with a DS-Fi2 digital camera was used in combination with the Nikon NIS-Elements Ar imaging software. The filter blocks DAPI and TRITC were used for co-localization with DAPI, Nile red and DASPMI. Additionally for the detection of GFP a Nikon GFP-L filter block (excitation 460–500 nm; emission >510 nm) was used.

Disaggregated cells were seeded at a density of 1 × 10⁵ cells/ml in 8 mL of complete RPMI 1640 medium and the formation of aggregates was examined at 72 hours of culture by phase contrast with a 10 × objective. To examine the effect of divalent cations on aggregate formation, cells were cultured in the presence or absence of 1 mM EDTA (Sigma-Aldrich) or EGTA (Sigma-Aldrich) for 24 h. For antibody-blocking experiments, cells were placed in 96-well culture plates at a density of 0.5–1 × 10⁶ cells/ml in 100–150 μl/well of complete RPMI 1640 medium containing 10 µg/ml anti-CD18 (Biolegend), anti-CD62L (BioLegend) mAbs or mouse IgG isotype control (BioLegend) and then incubated at 37°C for 2 h. Photomicrographs were taken using an ORCA-ER monochrome cooled-CCD camera (Hamamatsu, Hamamatsu, Japan) coupled to an Eclipse TE2000-E inverted fluorescence microscope (Nikon, Tokyo, Japan) and NIS-Elements AR imaging software and the number of aggregates containing more than 20 cells per field was counted.

Deparaffinized sections were boiled for 20 min in 10 mM citrate buffer (pH 6) (Genemed Biotechnologies) for antigen retrieval, rinsed in distilled water, and then blocked with 3% normal goat serum (Vector Laboratories) in PBS for 1 h at room temperature. The sections were incubated with primary antibodies in PBS or Can Get Signal immunostain solution (Toyobo, Osaka, Japan) for 1 h at room temperature. The primary antibodies were as follows: rabbit anti-GFAP, rabbit anti-Iba1, rabbit anti-NeuN, and rabbit anti-olfactory marker protein (OMP) (#ab183947, 1:1,000; Abcam, Cambridge, UK). After washing in PBS, the sections were incubated with Alexa Fluor 488- or 594-conjugated goat anti-rabbit IgG antibodies (#A31627 and #A-11012, 1:1,000; Thermo Fisher Scientific, Waltham, MA, USA) in PBS or Can Get Signal immunostain solution for 1 h at room temperature, washed in PBS, and mounted using VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories). All images were captured and analyzed using the ECLIPSE Ti confocal microscope (Nikon Instruments) with NIS-Elements AR imaging software version 4.00.06 (Nikon Instruments). The number of OMP-positive cells was measured in all bilateral subareas, and their mean values were calculated for each animal.