Pre-Embedding Labeling Methods

Abstract

Colloidal gold conjugates generally do not readily penetrate cells, even after permeabilization. Therefore, their use in pre-embedding immunostaining has been restricted to labeling cell-surface antigens for scanning (1) or transmission electron (Fig. 1A) microscopy or for tracing endocytic pathways in living cells (Fig. 1B). Recently, 1-nm gold conjugates that do penetrate cells and tissues much more readily have been used successfully to immunolabel intracellular structures (2, 3). For pre-embedding labeling, all of the immunostaining is done prior to embedding the tissue in resin or preparing the samples for scanning electron microscopy. This method is especially useful if the antigen to be detected is sensitive to fixation. The immunostaining may be done on unfixed or lightly (4% formaldehyde) fixed samples. Following the immunolabeling, the samples may then be refixed in 2% glutaraldehyde-2% formaldehyde to give good ultrastructural preservation.

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1 Introduction

Colloidal gold conjugates generally do not readily penetrate cells, even after permeabilization. Therefore, their use in pre-embedding immunostaining has been restricted to labeling cell-surface antigens for scanning (1) or transmission electron (Fig. 1A) microscopy or for tracing endocytic pathways in living cells (Fig. 1B). Recently, 1-nm gold conjugates that do penetrate cells and tissues much more readily have been used successfully to immunolabel intracellular structures (2, 3). For pre-embedding labeling, all of the immunostaining is done prior to embedding the tissue in resin or preparing the samples for scanning electron microscopy. This method is especially useful if the antigen to be detected is sensitive to fixation. The immunostaining may be done on unfixed or lightly (4% formaldehyde) fixed samples. Following the immunolabeling, the samples may then be refixed in 2% glutaraldehyde-2% formaldehyde to give good ultrastructural preservation.

When immunolabeling cell-surface components, it is important to be aware that redistribution of membrane components may be induced by the immunolabeling. Crosslinking of membrane components in unfixed or lightly fixed cells by antibodies may result in aggregation, capping, or internalization of the molecule of interest. Usually, brief fixation in a fixative containing low concentrations of glutaraldehyde is sufficient to prevent redistribution. Fixation in formaldehyde alone or performing the immunostaining at 4°C may not be sufficient to prevent lateral diffusion of molecules in the plasma membrane.

Fig. 1.

Transmission electron micrograph of RBL-2H3 cells with colloidal gold conjugated to a monoclonal antibody against the IgE receptor. (A) Gold conjugate is localized primarily in coated pits. (B) Five minutes after exposing cells to antibody-coated gold, gold particles are localized in early endosomes. Bar = 0.5 μm.

2 Materials

• 1. Dulbecco’s phosphate-buffered saline (PBS): 100 mg anhydrous calcium chloride, 200 mg potassium chloride, 200 mg monobasic potassium phosphate, 100 mg magnesium chloride 6H₂O; 8g sodium chloride, and 2.16g dibasic sodium phosphate 7H₂O; bring vol to 1L with deionized glass-distilled water, pH 7.4.

• 2. 0.5% Glutaraldehyde-2% formaldehyde fixative: 0.5 mL 70% EM-grade glutaraldehyde, 7mL 20% EM-grade formaldehyde, 3mg calcium chloride, and 62.5 mL 0.lM cacodylate buffer or PBS, pH 7.4 (see Note 1).

• 3. 0.lM Glycine: 75 mg glycine, bring the vol to 100 mL with PBS.

• 4. Dulbecco’s PBS without calcium and magnesium: 200 mg potassium chloride, 200 mg monobasic potassium phosphate, 8g sodium chloride, and 2.16g dibasic sodium phosphate 7H₂O; bring vol to 1L with deionized glass-distilled water, pH 7.4.

• 5. 1% Bovine serum albumin (BSA): 1g BSA and 100 mL PBS without calcium and magnesium; add BSA to PBS with stirring. Make fresh daily.

• 6. Primary antibody (see Note 2).

• 7. Colloidal gold conjugate (see Note 3).

• 8. 0.1M Cacodylate buffer: 21.4g cacodylic acid 3H₂O, sodium salt; bring vol to 1L with deionized glass-distilled water; adjust pH to 7.4 with HCl.

3 Methods

3.1 Cell-Surface Labeling

3.1.1 Unlabeled Primary Antibody

• 1. Rinse cells twice in PBS, and fix for 30 min to 1h at room temperature in 0.5% glutaraldehyde and 2% formaldehyde in PBS (see Note 4).

• 2. Following fixation, rinse the cells three to five times over a period of 30 min in PBS (see Note 5).

• 3. Rinse the cells for 5 min in 0.1M glycine in PBS to quench free aldehyde groups (see Note 6).

• 4. Rinse the cells for 15 min in a solution of 1% BSA in PBS without calcium and magnesium to block nonspecific binding of the primary antibody (see Note 7).

• 5. Expose the cells to the primary antibody. Incubation times usually range from l–2h at room temperature.

• 6. Rinse cells five times in PBS without calcium and magnesium over a period of 30 min to remove unbound primary antibody (see Note 8).

• 7. Incubate the cells in colloidal gold conjugate for 30 min at room temperature (see Note 3). 8.

• 9. For better morphological preservation, refix the cells in 2% glutaraldehyde-2% formaldehyde in 0.lM cacodylate buffer, pH 7.4 for 1h at room temperature.

• 10. Rinse in cacodylate buffer, postfix in l–2% osmium tetroxide, and embed as usual (see Chapter 36; Note 9).

3.1.2 Primary Antibody Conjugated to Colloidal Gold

• 1. Perform steps l–4 as outlined in Section 3.1.l.

• 2. Incubate the cells in primary antibody conjugated to colloidal gold diluted in PBS without calcium and magnesium containing 1% BSA for 1h at room temperature (see Note 2).

• 3. Continue with step 8 in Section 3.1.1.

3.1.3 Tracing Endocytic Pathways

• 1. Rinse cells in sterile medium containing l–2% BSA.

• 2. Add ligand conjugated to the colloidal gold, i.e., colloidal gold conjugated with antibody to a specific receptor.

• 3. Allow to bind at 4°C for 30–60 min, or return to incubator immediately.

• 4. At various time intervals, stop endocytosis with cold (4°C) PBS or balanced salt solution.

• 5. Rinse twice in cold PBS.

• 6. Fix and embed as usual (see Chapter 36; Note 9).

4 Notes

• 1. The purity of the reagents is critical. Always use the highest quality available, i.e., affinity-purified antibodies, EM-grade glutaraldehyde, distilled formaldehyde, or freshly prepared paraformaldehyde. The use of distilled formaldehyde, not formalin, which contains alcohol, is recommended. Freshly prepared paraformaldehyde can also be used, especially if large volumes of fixative are needed. To prepare an 8% solution of paraformaldehyde, in a fume hood add 2g of paraformaldehyde (trioxymethylene) powder to 25 mL of double-distilled water. With constant stirring, heat solution to 60–70°C. Once the solution has reached the proper temperature, continue to stir for 15 min. The solution will be milky. Add one to two drops of 1N NaOH, with stirring, until the solution clears. A slight milkiness may persist. Cool and filter through Whatman No. 1 filter paper. This solution should be used the same day that it is prepared.

• 2. Primary antibody is usually at a concentration of l–5 μg/mL, in PBS without calcium and magnesium, containing 1% BSA.

• 3. The colloidal gold conjugate should be directed against the primary antibody and diluted to l–5 μg/mL m PBS without calcium and magnesium plus 1%BSA.

• 4. The composition of the fixative may vary depending on the sensitivity of the antigen. For some antigens, it may be necessary to omit the glutaraldehyde entirely and fix in 4% formaldehyde in PBS for l–4h.

• 5. 0.lM Phosphate buffer, pH 7.4, may be substituted for PBS.

• 6. Following fixation in formaldehyde or glutaraldehyde, tissue is generally quenched. The purpose of this step is to block any free aldehyde groups that remain after fixation and washing. This step is especially critical after glutaraldehyde fixation. Glutaraldehyde is a bifunctional aldehyde. During fixation, one end may bind to cellular constituents, leaving the other end free to react. If this end is not blocked, it can bind to the protein in the blocking solution or to the primary antibody, increasing the nonspecific background. Although any small molecularweight compound containing an amino group may be used, the most commonly used quenching agents are glycine, ammonium chloride, and sodium borohydride.

• 7. Following queching, the samples are blocked to reduce nonspecific binding of the primary antibody and to reduce the background. The proteins in the blockmg solution are chosen so as not to react with the secondary antibody or detection system. They bind to sites in the samples that bind proteins through nonspecific interactions, such as charge. Although 2–10% BSA is most commonly used, other proteins, such as ova albumin, normal serum, or IgG fractions, can also be used. The exact composition of the blocking solution may have to be determined empirically if background staining is high.

• 8. If the nonspecific staining is high, 0.l M EDTA can also be added to the rinse solutions.

• 9. Appropriate controls should always be run with any immunocytochemical procedure. Controls may include omitting the primary antibody, substituting preimmune serum, normal serum, or normal IgG for the primary antibody, adsorbing the primary antibody against the antigen, or immunostaining with an unrelated antibody.