Fetuses were harvested on E18.5, and spleens were harvested from adult mice (7–16 weeks old). Tissues were immediately embedded in OCT, frozen on dry ice and stored at −80°C. Sections (8-μm) were cut at −20°C and then stored at −80°C until time of staining. At the time of staining, sections were removed from freezer and fixed in ice-cold acetone for 15 minutes, washed with PBS, and blocked for 10 minutes. Primary antibodies were applied overnight at 4°C and secondary antibodies were applied for 1 hour at room temperature. Coverslips were mounted using Prolong Diamond Antifade Mountant with Dapi (Fisher Cat# P36966). Images were taken using a Zeiss Axio Imager M2 Plus Wide Field Fluorescence Microscope. Antibodies used: CD4 (Biolegend catalog no. 100401), Madcam1 (R&D Biosystems catalog no. AF993), CD3 (Biolegend Cat# 100243), B220 (Biolegend catalog no. 103229), VCAM-1 (R&D Biosystems catalog no. AF643). Quantitation of staining intensity was performed using ImageJ and normalized to the area analyzed.
Axio imager m2 plus wide field fluorescence microscope
Manufactured by Zeiss
The Axio Imager M2 Plus Wide Field Fluorescence Microscope is a laboratory equipment product from Zeiss. It is a microscope designed for wide-field fluorescence imaging.
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2 protocols using axio imager m2 plus wide field fluorescence microscope
1
Immunohistochemistry of Mouse Spleen
2
Urothelial Cell Analysis in Mice
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WT and Ifrd1−/− mouse urine samples were collected for sediment analysis (10 μl urine plus 40 μl 1X PBS) and subjected to cytospin3. Sediments on the microscope slides were fixed in acetic acid/alcohol for 15 minutes and subjected to Epredia™ Papanicolaou EA Staining (22-050-211, Thermo Fisher Scientific, USA) following the manufacturer’s protocol. Bright field images from a whole slide scanner were used to identify and count the sloughed urothelial cells.
To assess for the production of ROS by the sloughed superficial cells, 50μl of urine was centrifuged to collect cells that were then incubated with 10μM dihydroethidium (ThermoFIsher Scientific, D23107) for 30 min at 37°C, rinsed in PBS, and then subjected to cytospin3. Subsequently they were mounted with Prolong Diamond Antifade Mountant with DAPI (ThermoFisher Scientific, P36962) and promptly imaged using Zeiss Axio Imager M2 Plus Wide Field Fluorescence Microscope.
To assess for the production of ROS by the sloughed superficial cells, 50μl of urine was centrifuged to collect cells that were then incubated with 10μM dihydroethidium (ThermoFIsher Scientific, D23107) for 30 min at 37°C, rinsed in PBS, and then subjected to cytospin3. Subsequently they were mounted with Prolong Diamond Antifade Mountant with DAPI (ThermoFisher Scientific, P36962) and promptly imaged using Zeiss Axio Imager M2 Plus Wide Field Fluorescence Microscope.
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