Nis element br 3

The left tibia from each rat was fixed with 4% paraformaldehyde in phosphate-buffered saline and decalcified with 10% ethylenediaminetetraacetic acid. Bone tissues were cut into thin sections (7 µm) and stained with H&E (Wako, Osaka, Japan). Stained sections were observed with a light microscope (NIS element BR 3.0; Nikon, Tokyo, Japan).

Fresh 4T1 cells were pre-cultured for 72 h with 50% fat-conditioned medium (derived from obese or control mice) in RPMI-1640 culture medium (GIBCO™), supplemented with 10% fetal bovine serum and 1% antibiotic and antimycotic (100 IU/mL penicillin and streptomycin—GIBCO™). 10⁵ cells were plated in RPMI-1640 without FBS in the upper chamber of a Transwell® plate (6.5 mm diameter and 8 μm pore size—Corning Incorporated Costar®). As a migratory stimulus, 400 ng/mL of each CXCL12 and CCL25 in RPMI-1640 without FBS was placed in the lower chamber. After 6 h, the membrane of the upper well was fixed and stained with Panotico kit (Laborclin). Cells were counted in a microscope using the NIS-Element BR 3.2 software (Nikon).

Astrocytes were fixed in methanol:acetone (1 : 1) for 20 min at −20 °C. After blocking, the cells were incubated overnight at 4 °C with chicken or rabbit GFAP, p-cPLA₂ antibodies (1 : 700, Dako, Carpinteria, CA, USA; 1 : 300, Abcam, Cambridge, MA, USA respectively). After washing three times in PBS, cells were labelled with anti chicken (594 nm, red) or rabbit (488 nm, green) Alexa Fluor secondary antibody (1 : 100, Life Technologies), for 1.5 h at RT. Following three PBS washes, cells were labeled with DAPI to visualize nuclei (1 : 800, blue, Life Technologies). All images were taken at ×200 magnification on Nikon Eclipse and processed by NIS-Element BR 3.2 software (Nikon Inc., Melville, NY, USA).

Stained sections were investigated with an inverted fluorescence microscope (Nikon TE2000; Nikon, Japan) equipped with a spot digital camera with three filters (for emission spectra maxima at 358, 461 and 555 nm). Both microscope and camera are connected to a computer system installed with image software (NIS Element BR-3.2; Nikon) including image merging and a fluorescence intensity analyzer. For laser confocal microscopy, the same microscope was used, which is equipped with a laser emission and detection system with three different channels. The optical scanning and image-processing tasks were run by the program Nikon EZ-C1 (v.3.80) including reconstruction of Z-stack images into projections or 3D images.

Stained sections were first investigated with an inverted fluorescence microscope (TE2000; Nikon, Tokyo, Japan) equipped with a spot digital camera with three filters (for emission spectra maxima at 358, 461, and 555 nm). Image-processing software (NIS Element BR-3.2; Nikon), including image merging and a fluorescence intensity analyzer, was installed on a computer system connected to the microscope. For laser confocal microscopy, we used the same microscope equipped with a three-channel laser emission system. SR-SIM [173 (link)] was performed using a Zeiss Elyra S.1 SIM system and a × 63/1.4 oil Plan-Apochromat objective (Zeiss, Oberkochen, Germany), sCMOS camera (PCO Edge), and ZEN 2012 software (Carl Zeiss Microscope). The resolution of the SR-SIM system at BioVis, Uppsala University, was 107 nm in the X–Y plane and 394 nm in the Z plane. The laser and filter were set up as follows: 405 nm laser of excitation coupled with BP 420–480 + LP 750 filter, 488 nm laser of excitation with BP 495–550 + LP750 filter, 561 nm laser of excitation with BP 570–620 + LP 750 filter, and 647 nm laser of excitation with LP 655 filter. From the SR-SIM dataset, 3D reconstruction was done with Imaris 8.2 (Bitplane, Zürich, Switzerland). A bright-field channel was able to merge fluorescence channels to visualize cell and tissue borders.

RNAscope^® sections were first investigated with an inverted fluorescence microscope (Nikon TE2000) equipped with a spot digital camera with three filters (for emission spectra maxima at 358, 461, and 555 nm). The image processing software was NIS Element BR-3.2 (Nikon). Laser confocal microscopy was performed using the same microscope equipped with a three-channel laser emission system. The software used was Nikon EZ-C1 (ver. 3.80). We used an Elyra S.1 SIM system with a 63x/1.4 oil Plan-Apochromat objective (Zeiss, Germany) and an sCMOS camera (pco. edge) with ZEN 2012 software. Multichannel SR-SIM imaging was conducted with a laser and filter setup: 405 nm laser of excitation coupled with BP 420–480 + LP 750 filter, 488 nm laser of excitation with BP 495–550 + LP750 filter, 561 nm laser of excitation with BP 570–620 + LP 750 filter, and 647 nm laser of excitation with LP 655 filter. SR-SIM images were processed using ZEN software. The 3D reconstruction was performed with Imaris 8.2 (Bitplane, Zurich, Switzerland). The resolution of the SIM system SR-SIM had a lateral precision of 80 nm (Gustafsson et al., 2008 (link)). The resolution was measured with subresolution fluorescent beads (40 nm; Zeiss) in the green channel (BP 495–550 + LP750). An average PSF value was obtained from multiple beads using the built-in experimental PSF algorithm of the ZEN software.