Dfc7000 t ccd camera

FISH was performed as described by Daims et al., [19 ]. Unless otherwise stated, a 3 h hybridisation period was determined to be sufficient for optimal fluorescent signal for the target group. Details about the optimal formamide concentration used for each probe are given in Table 1. The nonsense NON-EUB probe was applied to all samples as a negative control for sequence independent probe binding [20 (link)]. As a general stain for all microorganisms, 3.6 μM 4',6-diamidino-2-phenylindole (DAPI) was applied for 1 h at 4°C in the dark. Quantitative FISH estimations of target populations was performed using the DAIME software [21 (link)]. Biovolumes were calculated from 30 images acquired with a 63 x magnification objective lens. The abundance was measured as the percentage of the area fluorescing with the EUBmix [22 (link),23 (link)] and ARC915 [24 ] (both Cy5 labeled, collectively covering most Bacteria and Archaea) that also fluoresced with the target probe (Cy3). Microscopic analysis was performed with an Axioskop epifluorescence microscope (Carl Zeiss, Germany), equipped with a LEICA DFC7000 T CCD camera or a white light laser confocal microscope (Leica TCS SP8 X) (Leica Microsystems, Kista, Sweden).

The evaluations focused on the hippocampus. To analyse the gross morphology and neuronal cytoarchitecture of dentate gyrus (DG) and Cornus Ammonis (CA) hippocampal areas by light microscopy, H&E staining was performed as previously reported [39 (link),42 (link)]. Briefly, approximately 20 randomised sections (5 microscopic fields) per animal and time/condition were examined by a blinded operator using a Leica DM6B WF microscope (Leica Microsystems, Buccinasco, MI, Italy). The images were acquired with Leica dfc 7000 t CCD camera (Leica microsystems, Buccinasco, MI, Italy) and stored on a PC running the Leica Application Suite X (LAS X) software (Version 5.1.0).

Immunofluorescence experiments were performed as previously described [27 (link)]. Briefly, isolated tissues were fixed in 4% PFA solution at 4 °C overnight and then, after washing for 1 h in PBS, were immersed in 30% sucrose solution in PBS overnight. After washing in PBS, tissues were embedded in Tissue-Tek OCT compound (Sakura, The Netherlands) and frozen at − 80 °C. After blocking, sections were incubated with primary antibodies for 1 h at room temperature in blocking solution (0.1% Gelatin in PBS), washed for 30 min and then incubated with secondary antibodies for 1 h. Finally, the sections were washed for 15 min in PBS and mounted in PBS-glycerol (1:1) pH 8.0, containing 1% n-propyl gallate.
In the case of NMO-IgG staining, isolated tissues were frozen without fixation. Sections of 8 μm thickness were cut on a cryostat (CM 1900; Leica) at − 20 °C and stored on poly-l-lysine glass slides (positively charged) at − 80 °C. In this case, tissue sections were fixed at the end of the staining procedure to preserve conformational epitope integrity.
Finally, sections were viewed with a Leica DM2500 LED fluorescence microscope using 20X/0.55 and 40X/0.80 PL FLUOTAR objectives and photographed with a Leica DFC7000 T CCD camera. Confocal images were obtained using an automated inverted Leica TCS SP8 confocal microscope with a 100X HC PL Apo oil CS2 objective.