A length of 12 μm brain sections covering the entire arcuate nucleus were placed onto a series of slides with 4–5 sections on each slide. The distance between sections in single slide is 144–168 μm. One slide from each mouse that contains matched sections was used to compare controls and mutants. Zeiss Axio imager 2 with apotome was used to image in situ hybridization and immunohistochemistry result. Integrated density measurement in Image J software was used to analyze densitometry. For cell counting, analyze particles measurement in image J was used to count specifically immuno-stained cells in the arcuate nucleus.
Axio imager 2 with apotome
Manufactured by Zeiss
The Axio Imager 2 with Apotome is a microscope system designed for advanced imaging and analysis. It features a high-resolution Apotome optical sectioning module that enables optical sectioning and improved contrast in fluorescence imaging.
Automatically generated - may contain errors
4 protocols using axio imager 2 with apotome
1
Brain Section Analysis of Arcuate Nucleus
2
Multicolor Immunofluorescence Tissue Staining
3
Quantifying ISH/IHC Images in Embryos
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For quantification of ISH/IHC images, serial sectioning was performed on embryo/mouse brain with the distance between sections 84–216 μm. One slide from each mouse that contains matched sections was used to compare controls and mutants. Zeiss Axio imager 2 with apotome was used to image ISH and IHC results. Integrated density measurement in Image J software was used to analyze densitometry. For cell counting, specifically immuno-stained cells in the arcuate nucleus were counted. Quantifications were done by analyzing three rostral to caudal sections for each embryo/mouse and at least three embryos/mice per each experimental group. Statistical differences were determined by two-sided Student’s t-test. Statistical significance is displayed as follows: ns for not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
4
Immunofluorescent Analysis of Lymph Node Microenvironment
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LN, diaphragm, and colon were placed in O.C.T. compound (Tissue Tek) and frozen on dry ice. Blocks were cut into 5 µm sections on Superfrost/Plus slides (Fisher). Tissue sections were fixed in ethanol and acetone at a 1∶1 ratio, blocked in 3% BSA-1X PBS containing 10% donkey serum and Fc block (2.4G2). Sections were stained with biotin-anti-PD-L1 (BioLegend), Alexa488- or eFluor660-anti-Lyve1, eFluor450-anti-B220, eFluor450-anti-CD31, FITC- or biotin-anti-MAdCAM-1 (all from eBioscience). Secondary reagent used was Streptavidin-Dylight594 (Jackson Immunoresearch). Images were taken using an Axio Imager 2 with Apotome (Carl Zeiss), and modified by adjusting brightness and contrast to the same levels (Adobe photoshop). Using ImageJ 1.46 software, we established a threshold signal to define Lyve-1+ pixels and a selection gate was created for each LN location. This gate was then transposed onto PD-L1 or MAdCAM-1 images of the same slide and the MFI for these two markers was calculated for the Lyve-1+ pixel distribution.
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