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Preparation of Frozen Cells and Tissues for Immunolabeling
Various types of specimens are used in immunostaining procedures, including cell suspensions, cell lines grown on culture plates or glass slides, fresh or frozen tissues, curettings, biopsies, or whole-mounted organisms. Because cells and tissues are fragile, samples need to be prepared in ways that preserve the structural integrity of the cell during the immunostaining procedure. The following protocols are recommended to prepare fresh or frozen samples for immunostaining. Samples can be stored up to a week at -20 or -80°C in the freezer, but some cellular morphology may be degraded with time, so it is best to use them as soon as possible after sectioning. If the slides are to be shipped to another laboratory, they should be shipped on dry ice.
Preparation of Frozen Cell Smears using Cell Suspensions or Cultured Cells
Cell smears are useful for the detection of cell surface proteins because the cell membrane is no disrupted, preventing immunolabeling within the cytoplasm.
- Fill a staining dish with -20°C acetone and place in a -20°C freezer.
- For cells that are grown on culture plates, carefully remove the cells by manual scraping, trypsinization, or EDTA, and place them into a centrifuge tube. Cells that are suspended in media or plasma can be centrifuged directly.
- Centrifuge the tube at 1,000-3,000 RPM for 1 minute.
- Cool the tube on ice then carefully remove and discard the supernatant.
- Working quickly, smear a few microliters of cells onto a pre-cleaned, charged microscope slide.
- Air-dry the slide for a few minutes until it has just barely dried.
- Carefully place the slide into the acetone filled staining dish and incubate for 5 minutes at -20°C.
- Air-dry the slide at room temperature.
- Place the dried slide in a slide box and store at -20°C or -80°C.
Preparation of Frozen Cryosections using Cell Suspensions, Cultured Cells, or Tissues
Cryosections are useful for the detection of proteins throughout the cell because sectioning exposes the cytoplasm.
- Fill a staining dish with -20°C acetone and place in a -20°C freezer.
- For frozen tissues, trim to fit the tissue into the plastic cassette and proceed to step #8.
- For cells that are grown on culture plates, carefully remove the cells by manual scraping, trypsinization, or EDTA, and place them into a centrifuge tube. Cells that are suspended in media or plasma can be centrifuged directly.
- Centrifuge the tube at 1,000-3,000 RPM for 1 minute.
- Cool the tube on ice then carefully remove and discard the supernatant.
- Rinse the cell pellet once with PBS then repeat steps #4 and #5.
- Pipet the cells into plastic chuck.
- Add 1 ml of OCT embedding medium.
- Freeze until OCT medium is solid.
- Prepare cryosections of the OCT block using a cryostat.
- Dip the cryosections in -20°C acetone for 5 minutes.
- Air-dry the cryosections for 5 minutes at room temperature.
- Place the dried cryosections in a slide box and store at -20°C or -80°C.
Cryosections should be used as quickly as possible (within a few days); frozen sections can degrade with time, even when stored in the freezer. If cryosections need to be shipped to another laboratory, they should be boxed and shipped on dry ice.
For more information about LSBio Protocols contact Technical.Support@LSBio.com
