Axioskope microscope

For morphological analysis, cells were fixed for 20 min with freshly prepared 4% (w/v) paraformaldehyde in phosphate buffer saline (PBS), and stained with a primary antibody against Ionized calcium binding adaptor molecule 1 (Iba1) (rabbit, 1:250; #019-19741, Wako Pure Chemical Industries Ltd, Osaka, Japan), and a secondary antibody Alexa Fluor 594 goat anti-rabbit (1:1000; #R37117, Invitrogen Corporation, Carlsbad, CA, USA). To identify the total number of cells, microglial nuclei were stained with Hoechst 33258 dye (Sigma Chemical Co., St. Louis, MO, USA). Fluorescence was visualized using an AxioCam HRm camera adapted to an AxioSkope^® microscope (Zeiss, Germany). Pairs of U.V. and red-fluorescence images of 10 random microscopic fields (original magnification: 400×) were acquired per sample. To characterize microglia morphology we used the particle measurement feature in ImageJ (1.47v, USA) to automatically obtain the 2D area, perimeter, circularity, and Feret’s diameter of single microglia. Circularity of microglia was obtained by the formula: Circularity = 4π (area/perimeter²). A circularity value of 1.0 indicates a perfectly circular cell, and values near zero indicate elongated and ramified microglia. Feret’s (maximum) diameter, a measure of cell length, is the highest distance between any two points along the cell perimeter.

For morphological analysis, cells were fixed for 20 min with freshly prepared 4% (w/v) paraformaldehyde in phosphate-buffer saline (PBS) and a standard immunocytochemical technique was performed using a primary antibody raised against Iba-1 (rabbit, 1:250; Wako Pure Chemical Industries Ltd, Osaka, Japan), and a secondary Alexa Fluor 594 goat anti-rabbit (1:1000; Invitrogen Corporation, Carlsbad, CA, USA). To identify the total number of cells, microglial nuclei were stained with Hoechst 33258 dye. Fluorescence was visualized using an AxioCam HRm camera adapted to an AxioSkope^® microscope (Zeiss). Pairs of U.V. and red-fluorescence images of ten random microscopic fields (original magnification: 400×) were acquired per sample. To quantitatively characterize microglia morphology we used the particle measurement feature in ImageJ (1.47v, USA) to automatically measure the 2D area, perimeter, and Feret’s diameter of single microglia cells. Feret’s (maximum) diameter, a measure of cell length, is the greatest distance between any two points along the cell perimeter. We also evaluated the transformation index, first defined by Fujita et al. (1996) (link) as [perimeter of cell (μm)]²/4π [cell area (μm²)], which categorizes microglia ramification status. A cell with long processes and a small soma exhibits a large index that is dependent on cell shape but independent of cell size.