Provis ax70 microscope

Cells were seeded on coverslips and allowed to attach for 24 h. Cells were exposed to µG or maintained in standard conditions (NG) for 24 and 36 h. At the end of treatments, slides were washed with PBS, fixed in methanol, and incubated with DiOC6 for 45 min at 37 °C. Cells were then counterstained with DAPI. Photographs were taken with a Provis AX70 microscope (Olympus, Tokyo, Japan) and Cytovision software (Applied Imaging Corp., Santa Clara, CA, USA.) [26 (link)].

An indirect immunoperoxidase method using 7E11 or 6G11 as primary antibodies was performed on 5 μm-thick frozen tissue sections of each metastatic sample. The secondary antibody was a rabbit monoclonal anti-mouse IgG1 H&L (clone M1gG51-4, Abcam, UK) coupled with antirabbit OmniMap detection kit (Roche diagnostic, Meylan, France). The systematic controls used were absence of primary antibody and use of an irrelevant primary antibody of the same isotype.
For all tissue sections and for each antibody, cells expressing nANGPTL4 or cANGPTL4 were counted independently by two pathologists (AJ, CL) on five different fields at ×400 magnification. A ProvisAX70 microscope (Olympus, Tokyo) with wide-field eyepiece number 26.5 was used, providing a field size of 0.344 mm².
For each field, 100 tumor cells were analyzed. The percentage of nANGPTL4 or cANGPTL4-expressing cells was the number of positive cells among these 100 tumor cells. Results were expressed as mean ± standard error of the mean (SEM).
We were well aware that part of the staining was linked to antibody recognition of FLANGPTL4. However, the difference in number of cells expressing cANGPTL4 or nANGPTL4 on sister following tissue sections was necessarily linked to cANGPTL4 or nANGPTL4 fragment.

Serial sections (15-μm thick) from the hippocampus were cut on a cryostat LEICA CM1900 (Nussloch, Germany), mounted on gelatin-coated slides (n = 7 sections per slide) and stored at –20°C until further use. For immunolabeling, endogenous peroxidase activity was previously quenched with a solution of peroxide. After blocking with normal serum, sections were incubated overnight at 4°C with the primary antibody. A specific antibody against GFAP was used to detect astrocytes, anti-Iba-1 antibody was used to detect microglia, and an antibody against vascular cell adhesion molecule 1 (VCAM-1, for more details see Table 
1 and Additional file
1) was used to stain endothelial cells. Slides were incubated for 90 min at room temperature with the corresponding biotinylated secondary antibody. The signal was amplified with Vectastain ABC reagent (Vectastain ABC kit, Vector Laboratories, Burlingame, CA, USA) and the immunohistochemical stain was developed with 3,3′-diaminobenzidine. Slides were mounted with DePeX mounting medium (BDH, Poole, England) and photographed using an Olympus Provis AX70 microscope, coupled to an Olympus PD50 photography system. Image J software (Wayne Rasband, NIH, USA) was used to obtain the photographs and analyse the images.