Axioimager m2 fluorescent

Images of human brain sections were taken at 3 randomly selected locations within the grey matter of 1 brain section of the medial frontal gyrus. At the time of imaging, the investigator was blinded for the experimental group (aSAH or control). The sections were imaged with a 20× magnification on an epifluorescence microscope (Zeiss AxioImager M2 fluorescent microscope with N-Achroplan objectives, AxioCam MRm camera and Software Zen 2011). Images were evaluated and analyzed with the program ImageJ 2.0 (NIH, Bethesda, MD). For the microglia analysis (Iba-1 staining), the images were binarized using the triangle auto threshold algorithm in ImageJ. The percentage of the total area covered by Iba-1-immunopositive staining was measured. Particles larger than 100 pixels were considered as single cells. To analyze the activation of astrocytes, the intensity of the glial fibrillary acidic protein (GFAP) immunostaining was quantified by calculating the mean intensity per image. Furthermore, the percentage area of the GFAP-immunopositive area was quantified.

Imaging was performed in complete minimal medium supplemented with 2.5mM adenine and 2% glucose. Static images were obtained on a Zeiss Axio Imager M2 fluorescent light microscope with a 100x objective. Microfluidics were performed on a Zeiss Axio Observer Z1 using a CellAsics microfluidics plate with temperature controls and media flow of 2 psi on a Y0C4 yeast perfusion plate (channel size 3.5–5μm). Fluorescence intensity was analyzed using the Zen software package (Zeiss, Germany).