Brains harvested from mice were post-fixed overnight in 4% PFA, cryoprotected in 30% sucrose, processed for OCT embedding and sectioned at 40 μm as previously described [33 (link)]. For immunohistochemistry, standard methods with antigen retrieval in sodium citrate were used [34 (link)]. Antibodies used in this study included: anti-Olig2 (Millipore 1:500), anti-myelin basic protein (MBP; Millipore 1:500), anti-Cleaved Caspase 3 (CC3; Cell Signaling 1:500), and anti-Nkx2.2 (Iowa Hybridoma 1:50). Immunoreactivity was detected by incubation with appropriate Alexa-conjugated secondary antibodies (Molecular Probes—Life Technologies). Images of brain sections were captured using a Nikon NIE fluorescent microscope. The number of immune-positive cells per 100 μm2 area was quantified using Nikon Elements analysis software. Statistical significance was determined using Student’s t-test and presented as mean cells per field. All studies were blinded and performed on coded sections (n = 6 hypoxic + 6 normoxic animals for IHC and Western blotting (each)).
Ni e fluorescent microscope
Manufactured by Nikon
The Ni-E fluorescent microscope is a high-performance microscope designed for fluorescence imaging. It provides precise control over optical parameters and offers a range of advanced features to support various scientific and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Bx60 f5 microscope, by Leica (2 mentions)
Application suite software version 4, by Leica (2 mentions)
Dfc310 fx camera, by Leica (2 mentions)
Elements analysis software, by Nikon (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti olig2, by Merck Group (1 mentions)
Alexa conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Nis elements software, by Nikon (1 mentions)
4 protocols using ni e fluorescent microscope
1
Mouse Brain Preparation and Immunohistochemistry
2
Hypothalamic Nuclei pSTAT Analysis
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The Rat Brain Atlas by Paxinos was used to appropriately match hypothalamic levels −1.3 to −3.30 mm caudal to bregma. Images were taken using an Olympus BX60 F5 microscope with a Leica DFC310 FX camera and Leica Application Suite software version 4.6 (Leica Microsystems, Buffalo Grove, IL). Positively-stained cells were identified in the ventromedial hypothalamus (VMH), the arcuate nucleus (ARC), and the paraventricular nucleus of the hypothalamus (PVH). Average numbers of pSTAT positive cells across each nucleus were obtained. An individual blinded to the experimental treatment groups scored the sections. Publication quality pSTAT photos were taken using a Nikon Ni-E fluorescent microscope (Melville, NY).
3
Quantifying Amyloid Plaque Burden
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For each mouse, three slices surrounding the injection site were imaged at 10x magnification on a Nikon Nie Fluorescent Microscope and analyzed using Nikon NIS-Elements software. Each hippocampus was treated as an independent region of interest (ROI), and each CA1 region was also treated as an independent ROI. Plaque counting and size analysis was performed blinded on randomized slides and Look Up Tables (LUTs) between each image were set identically (50 min, 850 max). Only objects larger than 5 pixels (2.37 μm2) and less than 1500 μm2 were included in the analysis to reduce artifacts. Plaque burden was calculated as % plaque area / % ROI area. Since 3 slices were used per mouse, and each (independently injected) hippocampus was treated as an independent ROI, we obtained an n=30 for the proSAAS group and n=24 for the eGFP control group (due to the exclusion of one mouse, as described above).
4
Quantifying Hypothalamic Nuclei pSTAT
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