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Light Microscopic Immunocytochemistry Protocol For Tissue Culture
Most of these procedures have been developed by Dr. David M. Sherry of the University of Houston and graciously supplied to Signature Immunologics. We at Signature Immunologics have modified them specifically to address use of our IgGs in detecting amino acids. These protocols are similar to HPI protocols, but some details heve been changed to meet the requirements of working with isolated cells in polystyrene dishes. The Sherry laboratory uses modified 30 mm polystyrene dishes. A circle is drilled out of the bottom of the dish and a glass coverslip is attached to the bottom with Sylgard® (Dow-Corning), creating a well. The well requires 50 Ál of reagent to cover it completely, thus minimizing the amount of IgG used.

As basic vehicle, use a PBS formulation osmotically proper for cultured cells, as they lack structural support from other cells or an embedding matrix. Care must be taken to ensure that liquid exchanges do not wash the cells off the dish. This can occur at any point in the procedure from fixation to coverslipping. Gentle fluid transfer is sufficient for washes. Composition of the fixation buffer can also impact adhesion if it strongly buffers divalent cations. Do not allow the well to dry out at any time as this can raise backgrounds to unacceptable levels. Triton X-100 (0.1%) is added to the primary antiserum diluent to enhance antibody penetration. While it may not be necessary for all IgGs, it tends to provide a very constant level of performance across preparations and certainly does not attenuate signals.

Immunofluorescence is the preferred detection method for cultured cells; it is much less prone to artifactual labeling than enzyme-linked techniques and is more quantitative. Enzyme-linked methods can be very sensitive but have very narrow dynamic ranges, with poor differentiation of amino acid levels in different cell types. Amino acid detection for molecules such as glutamate, aspartate, glutamine, glycine, etc. are not all-or-none procedures as cells will contain varied levels that may discriminate functional cell types. The most quantitative method is silver-enhancement, but this requires additional steps (see Resources ).

Culture dishes labeled with fluorophores can be coverslipped in an anti-fade DABCOtm, IMMUMOUNTtm mountant and stored under conditions optimal for various fluorophores (FITC< 0°C, TRITC 4°C). Background signals may be slightly higher with in vitro experiments compared to post-embedding sections: Agents that may normally bind antisera non-specifically may be eliminated by embedding and etching procedures; coverglasses in culture systems coated with adhesion agents may increase background; and, with in vitro labeling, the specimen is 5-10 um thick compared to 500 nm less for most postembedding sections. The Sherry laboratory prefers FITC visualization because of the long-term stability of FITC under low temperature storage. We at Signature Immunologics have developed a preference for Cy3tm (Pharmacia-Amersham) for quantitative imaging because it fluoresces at wavelengths better matched to CCD cameras, has a lower rate of photobleaching and high quantum efficiency.

    Protocol
  1. Fixation: Quantitative trapping of free amino acids requires a bifunctional linker such as glutaraldehyde. Optimal trapping will occur with a standard 0.5%-2.5% glutaraldehyde/1% paraformaldehyde mixture. Depending on the amount of native antigen in cells, even lower levels of glutaraldehyde may be used along with higher paraformaldehyde (see Resources). The increased path length through cultured cells often permits low levels of glutaraldehyde to yield excellent signals. Fix at 4 hrs-overnight at 4°C.
  2. Wash 10 min 3X in PBS
  3. Reduce autofluorescence and glutaraldehyde induced fluorescence: Wash 1 min in 0.5% NaBH4 in deionized water (not PBS). Note: This step could mechanically damage cells if left too long. This step is imperative if FITC or or any other green dominant fluorophore is used, but is less critical (though still beneficial) for long-wavelength fluorophores such as rhodamine or Cy3tm (Amersham).
  4. Wash 10 min 3X in PBS. Carefully dry around the well with a lint-free wipe.
  5. Primary Blocking: Blocking is usually not essential with Signature Immunologics reagents in post-embedding formats but may be needed with cultured cells. If desired, block with 50 Ál of 2% goat serum / 0.1% Triton X-100 PBS for 1 hr.
  6. Apply antiserum: Remove blocking agent with a pipet and replace with 50 Ál of Signature Immunologics IgG diluted with GSPBS + 0.1% Triton X-100. Incubate overnight at room temperature or 4°C if desired. The recommended dilutions should work well for most amino acids, but should signals seem weak, 1.5-2X stronger dilutions be used. However, if much higher concentrations are required it is very likely that (a) there is simply to little amino acid to detect; (b) fixation losses have been excessive; (c) the detection method is insensitive.
  7. Wash 10 min 3X in PBS. Carefully dry around the well.
  8. Secondary Blocking: If desired, block with 50 microliters of 2% GSPBS / 0.1% Triton X-100.
  9. Secondary antiserum: Incubate 30-45 min in secondary goat anti-rabbit IgG diluted as recommended by the manufacturer and supplemented with 0.1% Triton X-100.
  10. Wash 10 min 3X in PBS plus 5 min X4 in deionized water. Dry around well.
  11. Silver intensification (If using fluorescence detection, skip to the next step): Intensify as specified in Resources.
  12. Coverslip: Fill well with antifade mountant such as IMMUMOUNTtm, DABCOtm or, for FITC, 1 part PBS, 3 parts glycerol, plus 2.5% Na azide. A coverslip can be placed over the well if desired. If the cells are to be used for confocal microscopy, the coverslip containing the cells is carefully removed with a razor blade and mounted on a slide with antifade mountant. Silver intensified specimens may be permanently mounted in standard media.
  13. Storage: Store FITC frozen. Other fluorescent labels may be stored at 4°C or frozen as appropriate for the fluorescent tag. Silver images are archival.

Disclaimer: As conditions of use are outside our control, Signature Immunologics makes no warranties, express or implied, and assumes no liability in connection with use of this protocol.

© 2000 Signature Immunologics, Inc.
Copying and distribution: This Technical Note may be copied and distributed for educational and non-profit
purposes as long as acknowledgement of Signature Immunologics' copyright is included
in every instance of use. It may not be sold or distributed for commercial gain.

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