FFPE samples were preprocessed on a prototype S2 Singulator system. The sample was automatically processed in a NIC+ cartridge (S2 Genomics #100-215-389) by three 15 minute deparaffinization steps (CitriSolv, VWR), rehydrated by successive 1 mL washes of 100%, 100%, 70%, 50%, and 30% ethanol, followed by 2 washes of PBS. The sample was then spun at 1,000g for 3 min and resuspended in 0.5 mL Nuclei Isolation Reagent (NIR, S2 Genomics, #100-063-396) with 0.1 ul/uL RNase inhibitor (Protector, Millipore Sigma, #3335399001); all subsequent solutions had RNase inhibitor. The sample was dissociated to single nuclei in a second NIC+ cartridge with 2 mL of NIR for 10 min followed by a 2 mL wash with Nuclei Storage Reagent (NSR, S2 Genomics, #100-063-405). The single nuclei suspension was spun 500g for 5 min, resuspended in NSR, and counted.
Citrisolv
Manufactured by Avantor
Citrisolv is a concentrated, water-soluble cleaning agent designed for laboratory applications. It is formulated to effectively remove a variety of contaminants, including organic materials, from laboratory glassware and equipment.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse ti fluorescence microscope, by Nikon (1 mentions)
Image pro 10, by Media Cybernetics (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Aluminum potassium sulfate, by Merck Group (1 mentions)
Carmine, by Merck Group (1 mentions)
Carmine alum, by Merck Group (1 mentions)
Citrisolv, by Thermo Fisher Scientific (1 mentions)
Cytoseal, by Avantor (1 mentions)
4 protocols using citrisolv
1
Automated FFPE Nuclear Isolation
2
Pentachrome Staining for Osteoid Matrix
3
Fluorescence Imaging of Intestinal Tissue
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The small intestines (duodenum, jejunum, and ileum) from sham and VSG control mice were paraffin-embedded and sectioned onto slides by the University of Michigan In-Vivo Animal Core. Paraffin was removed using Citrisolv (VWR). The slides were incubated overnight at 4°C with primary antibody (see above). The slides were stained with corresponding secondary antibodies conjugated to fluorescence dyes (see above) for 2 hours at room temperature. The slides were mounted in an antifade fluorescence mounting medium containing DAPI (VECTASHIELD with DAPI, Vector Laboratories, catalog H-1200). Fluorescence images were obtained using an Olympus IX73 inverted fluorescence microscopy system or Nikon Eclipse Ti fluorescence microscope with a motorized X-Y stage system. Images were analyzed using Olympus cellSens imaging software or Image Pro 10 software (Media Cybernetics).
4
Whole Mount Tissue Preparation
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X-gal stained whole mounts were post-fixed in 4% PFA, washed twice with 1X PBS, dehydrated through an increasing ethanol gradient, cleared of lipids in Carnoy’s Fixative (60% Ethanol, 30% Chloroform, 10% Glacial Acetic Acid) for 2 h, and further cleared in Citrisolv (Fisher Scientific, Suwanne, GA) for 2 h. Glands were pressed flat between the slide and coverslip under a heavy weight for 30 min, and imaged on a Leica dissecting microscope Model WILD M3Z (Leica Microsystems, Bannockburn, IL) with an Optronics digital camera Model 60800 (Goleta, CA). The glands were then re-hydrated through a decreasing ethanol gradient and counterstained with Carmine alum (500 mL distilled water containing 1 g Carmine and 2.5 g aluminum potassium sulfate; Sigma Aldrich, St Louis, MO) diluted 1:4 in distilled water. Glands were once again dehydrated in ethanol, cleared in Carnoy’s Fixative and Citrisolv, and pressed flat before mounting under a coverslip with Cytoseal (VWR, West Chester PA) then re-photographed.
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