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"The following protocol has been developed and optimized by R&amp;amp;D Systems IHC/ICC laboratory for ICC experiments using cells grown on gelatin-coated glass coverslips.\r\n\r\nThis protocol provides a basic guide for the preparation, fixation, and fluorescent staining of cells on glass coverslips. Each investigator must determine the precise experimental conditions required to generate a strong and specific signal for each antigen of interest. If R&amp;amp;D Systems primary antibodies are employed, please refer to the product data sheets to obtain the recommended working dilutions. In the staining protocol, signal visualization is achieved using R&amp;amp;D Systems NorthernLights\u2122 range of fluorescent secondary antibodies and reagents. For all other reagents, please follow the manufacturer\u2019s instructions.\r\n\r\nPlease read the protocol in its entirety before starting.\r\n\r\nProtocol for Coverslip Preparation Using Gelatin\r\n\r\nMany cultured cell types do not adhere well to glass coverslips. Adding a thin layer of gelatin to a coverslip enhances the adhesion of cultured cells to glass.\r\n\r\n\r\nReagents Required\r\n\r\n\r\n\tGelatin-coating solution: 0.1% gelatin in deionized H2O\r\n\r\n\r\nMaterials\r\n\r\n\r\n\tCoverslips (sterilized)\r\n\tCell culture plate (6- or 24-well)\r\n\r\n\r\n\r\nProcedure\r\n\r\n\r\n\tPlace sterilized coverslips into the wells of a 24-well plate.\r\n\tAdd 400 \u00B5L of the gelatin-coating solution, and incubate the coverslips for 10 minutes at room temperature.\r\n\tRemove the gelatin-coating solution, and air dry the coverslips for 15 minutes.\r\n\tThe dried coverslips can now be stored at room temperature until use.\r\n\r\n\r\nProtocol for the Preparation &amp;amp; Fixation of Cells on Coverslips\r\n\r\n\r\n\r\nView Full Protocol\r\n\r\nReagents Required\r\n\r\n\r\n\t1X PBS: 0.137 M NaCl, 0.05 M NaH2PO4, pH 7.4\r\n\tFormaldehyde Fixative Solution: 2-4% paraformaldehyde in PBS\r\n\tWash buffer: 0.1% BSA in 1X PBS\r\n\tCell culture medium\r\n\r\n\r\nMaterials\r\n\r\n\r\n\tGelatin-coated coverslips in a 24-well plate\r\n\r\n\r\n\r\nProcedure\r\n\r\n\r\n\tCulture cells by adding 500 \u00B5L of culture media containing approximately 5000 cells to the wells of a cell culture plate containing gelatin-coated coverslips.\r\n\tWhen cells have reached the desired density/age, remove the culture media from each well and wash twice with PBS.\r\n\tAdd 300-400 \u00B5L of 2-4% Formaldehyde Fixative Solution to each well, and incubate for 20 minutes at room temperature.\r\n\tNote: Some cell types can be damaged by the change in surface tension that occurs when the culture medium is entirely removed and replaced with wash buffer. If this is the case, pre-fix the cells by adding 500 \u00B5L of 4% Formaldehyde Fixative Solution directly into the culture medium. After 2 minutes, replace the pre-fixation culture medium with 300-400 \u00B5L of 2% Formaldehyde Fixative Solution and incubate, for 20 minutes at room temperature.\r\n\tWash the wells twice with PBS and cover with 400 \u00B5L of wash buffer. The coverslips can be stored at 2-8 \u00B0C for up to 3 months or they may be stained immediately.\r\n\tNote: Fixation can result in hydrophobic cross-linking of tissue proteins. The time, temperature, pH, and fixative used will determine the degree of cross-linking. Once the fixation protocol has been optimized, the same procedure should be used consistently.\r\n\r\n\r\nProtocol for the Fluorescent ICC Staining of Cultured Cells on Coverslips\r\n\r\nThis protocol has been developed and optimized using R&amp;amp;D Systems NorthernLights fluorescent secondary antibodies but can be modified accordingly.\r\n\r\n\r\n\r\nView Full Protocol\r\n\r\nReagents Required\r\n\r\n\r\n\tPrimary Antibodies\r\n\tBlocking buffer: 10% normal donkey serum, 0.3% Triton\u00AE X-100\r\n\tDAPI (4',6-diamidino-2-phenylindole) solution: Add 1 \u00B5L of 14.3 mM stock for every 5 mL of PBS. Store any unused DAPI at 2-8 \u00B0C, wrapped in aluminum foil\r\n\tDeionized H2O\r\n\tDilution buffer: 1X PBS, 1% bovine serum albumin (BSA), 1% normal donkey serum, 0.3% Triton X-100, and 0.01% sodium azide\r\n\tAnti-fade mounting medium\r\n\tNorthernLights-conjugated secondary reagents (or equivalent)\r\n\t1X PBS: 0.137 M NaCl, 0.05 M NaH2PO4, pH 7.4\r\n\tWash buffer: 0.1% BSA in 1X PBS\r\n\r\n\r\nMaterials\r\n\r\n\r\n\tCell-covered coverslips in a 6- or 24-well plate\r\n\tFine tweezers\r\n\r\n\r\n\r\nProcedure\r\n\r\nNote: This protocol is optimized for cells grown on coverslips in a 6- or 24-well plate but can be adapted accordingly.\r\n\r\n\r\n\tWash the coverslips containing the fixed cells two times in 400 \u00B5L of wash buffer.\r\n\tBlock non-specific staining by adding 400 \u00B5L of blocking buffer and incubate for 45 minutes at room temperature.\r\n\tRemove blocking buffer. No rinsing is necessary.\r\n\tDilute the unconjugated primary antibody (or fluorescence-conjugated primary) in dilution buffer according to the manufacturer\u2019s instructions. For fluorescent ICC staining of cells on coverslips using R&amp;amp;D Systems antibodies, it is recommended to incubate at room temperature for 1 hour. Alternatively, incubate overnight at 2-8 \u00B0C.\r\n\tNote: Appropriate controls are critical for the accurate interpretation of IHC/ICC results. All IHC/ICC experiments should include a negative control using the incubation buffer with no primary antibody to identify non-specific staining of the secondary reagents. Additional controls can be employed to support the specificity of staining generated by the primary antibody. These include absorption controls, isotype matched controls (for monoclonal primary antibodies), and tissue type controls.\r\n\tWash two times in 400 \u00B5L of wash buffer. If using a primary antibody with a direct fluorescent conjugate, go to step 8.\r\n\tDilute the secondary antibody in dilution buffer according to the manufacturer\u2019s instructions. Add 400 \u00B5L to the wells, and incubate at room temperature for 1 hour in the dark. From this step forward samples should be protected from light.\r\n\tNote: R&amp;amp;D Systems NorthernLights fluorescent secondary antibodies and streptavidin conjugates are bright, resistant to photobleaching, and are ideal for multi-color fluorescence microscopy.\r\n\tNote: If a biotinylated antibody was used in step 4, apply the appropriate NorthernLights Streptavidin conjugate in step 6.\r\n\tRinse two times in 400 \u00B5L of wash buffer.\r\n\tAdd 300 \u00B5L of the diluted DAPI solution to each well, and incubate 2-5 minutes at room temperature. DAPI binds to DNA and is a convenient nuclear counterstain. It has an absorption maximum at 358 nm and fluoresces blue at an emission maximum of 461 nm.\r\n\tNote: DAPI counterstain can obscure visualization of targets localized in cell nuclei.\r\n\tRinse once with PBS and once with water.\r\n\tCarefully remove the coverslips from the wells and blot to remove any excess water. Dispense 1 drop of anti-fade mounting medium onto the microscope slide per coverslip. Mount the coverslip with the cells facing towards the microscope slide.\r\n\tVisualize using a fluorescence microscope and filter sets appropriate for the label used. Slides can also be stored in a slide box at &amp;lt; -20 \u00B0C for later examination.\r\n\tNote: Initial IHC/ICC studies often require further optimization and/or additional troubleshooting steps.\r\n\r\n\r\n\r\nTriton is a registered trademark of Dow Chemical.\r\n"^^<http://www.w3.org/2001/XMLSchema#string>
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