Icc50 hd

To verify the accuracy of the lesion, rats were deeply anaesthetized with sodium pentobarbital and perfused transcardially with 4% paraformaldehyde in .1 M phosphate buffer. Brains were immediately removed and stored in the fixation solution at a temperature of 5°C. Coronal sections of the NAcSh were cut at a thickness of 50 μm using a vibratome (Leica, Germany) and stained with cresyl violet. Pictures of the NAcSh (Figure 2b) were taken with the microscope Leica DM750 at two magnifications (4× and 10×) and the camera Leica ICC50 HD. Percentage of lesioned areas was calculated using ImageJ (1.53k) software by manually outlining the area with neurotoxic damage. Exclusion criteria were unilateral or misplaced lesions. One rat in the NAcSh lesioned group was excluded due to the wrong location of the lesion, another one due to unilateral lesion and a third one due to a unilateral extension of the lesion to bordering areas.
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We followed the method described by Vidoz et al. (2016) . Hypocotyl sections of control, flooded and AgNO3-treated flooded plants, were fixed in FAA (10 formaldehyde 40%: 5 acetic acid: 50 ethanol: 35 water v/v) by generating vacuum for 15 min. Samples were sequentially dehydrated with tertiary butyl alcohol series and finally embedded in paraffin. Finally, 30 µm sections were obtained with a microtome, stained with safranine for 2 h and successively observed with an optical microscope (Leica DM LB2, Leica Microsystems). Three biological replicates were processed for each treatment and 20 sequential sections for each replicate were observed. Representative pictures were then acquired with a digital camera Leica ICC50 HD. These sections were used to obtain measurements of cortex cell area, cortex cell density and area of cortex cells with the open source software ImageJ (NIH, http://rsb.info.nih.gov/ij).

Ten zebrafish larvae, in triplicate, randomly collected at each sampling time from the different tanks, were fixed by immersion in 4% paraformaldehyde and stored at 4°C for 24 h. Larvae were washed three times with PBS 0.1 M (pH 7.4) buffer for 10 min and preserved in ethanol (70%). Larvae were then dehydrated by subsequent washing in ethanol (80%, 95%, and 100%), washed with clearing agent ''Histo-Clear'' (Bio-Clear, Bio Optica) and embedded in liquid paraffin (Bio-Optica, Milano, Italy) at 55-58°C. Solidified paraffin blocks were cut with a microtome (Leica RM2125 RTS) to obtain 5 lm sections then stained with Hematoxylin (Mayer) and Eosin Y (Sigma-Aldrich). Sections were observed using a Leica MD750 optical microscope connected with a camera Leica ICC50 HD.