Postembedding Immunogold Labeling

For postembedding immunocytochemistry, it is preferable to collect thin sections of Lowicryl embedded samples on Formvar-coated nickel slot grids. Nickel grids are inert in solutions used for immunostaining, whereas slot grids provide a relatively large viewing area void of support mesh that can obscure regions of interest. Numerous protocols for postembedding immunocytochemistry have been published, for a complete review see refs. 29 and 30. The following has proven successful for labeling cytoplasmic, structural, and membrane-associated proteins following the previously mentioned fixation protocol.

The following layout can accommodate the staining of 10-15 grids per run. For each wash step, dispense approx 50-100 ^L of solution per grid on a Parafilm sheet: to wash 10 grids spot three rows of 10 solution drops on a sheet of Parafilm approx 10 x 20 cm. It is advisable to filter the wash solutions before use to decrease the accumulation of "dust" particles on the sections. Furthermore, it is beneficial to remove as much solution as possible between changes of solution type, this can be aided by briefly dabbing the edge of the grid on filter paper before placing it in the next solution drop. For incubations of long duration, such as with antibodies, it is advisable to use a humidified chamber to

Caenorhabditis Elegans

Fig. 3. Immunogold labeling of Caenorhabditis elegans tissues fixed with potassium permanganate and embedded in Lowicryl. (A) The dorsal nerve cord of an adult hermaphrodite before immunostaining; the section thickness is 50 nm. Bar = 500 nm. (B) A 50-nm section stained with a primary antibody directed against SYD-2 (19), a protein required for presynaptic differentiation (34), diluted 1:50, revealed by 10 nm gold-conjugated secondary antibodies diluted 1:50. Notice the decrease in membrane contrast when compared to A. This is likely owing to extraction of KMnO4 during immunostaining. Bar = 200 nm. (C,D) Examples of immunostaining within the excretory canal and intestine using a primary antibody directed against the B-subunit of the v-type H+-ATPase (35) diluted 1:50 and revealed by 10 nm gold-conjugated secondary antibody diluted 1:50. Bar = 200 nm.

Fig. 3. Immunogold labeling of Caenorhabditis elegans tissues fixed with potassium permanganate and embedded in Lowicryl. (A) The dorsal nerve cord of an adult hermaphrodite before immunostaining; the section thickness is 50 nm. Bar = 500 nm. (B) A 50-nm section stained with a primary antibody directed against SYD-2 (19), a protein required for presynaptic differentiation (34), diluted 1:50, revealed by 10 nm gold-conjugated secondary antibodies diluted 1:50. Notice the decrease in membrane contrast when compared to A. This is likely owing to extraction of KMnO4 during immunostaining. Bar = 200 nm. (C,D) Examples of immunostaining within the excretory canal and intestine using a primary antibody directed against the B-subunit of the v-type H+-ATPase (35) diluted 1:50 and revealed by 10 nm gold-conjugated secondary antibody diluted 1:50. Bar = 200 nm.

prevent the evaporation of the small volumes of liquid. A humidified chamber can be easily assembled by placing damp paper towels around the periphery of the Parafilm sheet and enclosed by placing an inverted plastic box over both.

1. Incubate the grids in 0.05 M glycine in PBS for 15 min (optional; see Note 21).

2. Move the grids into Aurion blocking solution and incubate for 30 min. This blocking solution decreases nonspecific antibody interactions.

3. Wash three times in PBS; incubate for 5 min each time.

4. Incubate for 1 h in diluted primary antibody.

5. Wash six times in PBS; incubate for 5 min each.

6. Incubate for 1 h in gold-conjugated secondary antibodies diluted 1:50-1:200.

7. Wash nine times in PBS; incubate for 5 min each.

8. Fix the antibody conjugates by incubating in 2% gluteraldehyde in PBS.

9. Wash once in PBS for 5 min.

10. Wash twice in deionized water for 5 min each.

11. Allow the grid to dry before viewing in the electron microscope. It is advisable to counterstain the sections with uranyl acetate and lead citrate before viewing as much of the KMnO4 contrasting agent can be extracted during immunostaining (see Fig. 3B-D and Note 22).

4. Notes

1. At the moment, two commercially available high-pressure freezers are typically used for freezing: the Leica EMPact (Leica) and the BAL-TEC HPM 010 (BAL-TEC). The Leica model offers several benefits: its compact size, its mechanism for handling frozen samples (an automated ejection arm), the ability to use different types of freeze chambers to accommodate various sample types, a graphical readout of freezing conditions as the sample is being frozen, lower liquid nitrogen consumption rates, and its price. However, the author had difficulty obtaining adequately frozen samples using the current version of the Leica EMPact. Therefore, the methods described in this chapter have been performed with the BAL-TEC.

2. Besides Leica, BAL-TEC offers a commercially available automated substitution system, the FS 7500 freeze substitution system, which provides regulated control over temperature and temperature gradients. An alternative substitution system can be assembled from common lab items: a Styrofoam box, an aluminum block, a thermocouple, and dry ice. For further details see ref. 31.

3. The goal of freeze substitution is to remove water from the frozen sample so that ice crystallization will not cause damage when the sample is warmed; therefore, it is critical to use anhydrous solvents for this step. However, there have been reports of successful substitutions when small amounts of water are present in the solvent (32).

4. Commercial tannic acids are a mixture of polyphenolic compounds and vary considerably between manufactures. For the desired staining of membranes, the correct concentration of tannic acid for each manufacturer or even each lot may need to be determined empirically.

5. Oxygen inhibits the polymerization of Lowicryl. When handling the solution try not to introduce excess air by repeat pipetting or stirring. Bubbling nitrogen gas through the solution can remove excess oxygen.

6. Immunoelectron microscopy is not the technique to use to characterize antibodies; it is best to use well-characterized antibodies. The optimal antibody dilution is, of course, antibody specific. As a starting point, dilute the antibody roughly

10-fold less than you would for in situ staining or Western blotting. For double labeling experiments, two antibody sera from two different species can be mixed and used as a primary staining cocktail followed by staining with a cocktail of two different sized gold-conjugated secondary antibodies, one size specific for each primary antibody.

7. Air pockets within the freeze chamber act as insulation, thus, decreases the cooling rate within the sample. In addition, these pockets can collapse under high pressure, causing damage to the freeze chamber and sample. Therefore, it is imperative to fill the freeze chamber completely with material. Because excess water increases the chance of ice damage, it is preferable to use a filling material with relatively low water content and that will not adversely affect the physiology of the animals and introduce artifact. In this protocol, week-old E. coli is used as a fill for two reasons: its water content is relatively low and it is a medium that the animals usually encounter when reared in the laboratory. However, other materials have been used as fill, such as, yeast paste (7), agarose (12), and 1-hexadecene (18).

8. Removing excess bacteria from the freeze chamber rim aids in preventing the animals from swimming out of the chamber. When the nose of an animal contacts the metal, the animal typically reverses direction.

9. The number of animals loaded into the freeze chamber can vary depending on the type of experiment. For example, if embryos are of interest, the freeze chamber can accommodate many; for freezing adults, place approx 10-15 animals, just following the L4 to adult molt, into the freeze chamber. When loading the sample, try to be gentle so that artifact is not introduced due to mechanical damage.

10. Frozen samples can be stored for long durations in liquid nitrogen. One way to do this is to place the frozen sample into a Nalgene® cryotube in which a few small holes have been drilled—the holes allow nitrogen gas to escape; therefore, tubes remain submerged. The tubes can be moved into a cryostorage box and stored in a liquid nitrogen storage tank.

11. This particular mesh vial allows access to the frozen sample, which is important when removing the sample from the freeze chamber at low temperatures before embedding. To track the identity of each sample, one can place a notch in the wall of one vial and place it in the 12 o'clock position within the universal container, then note which sample is placed in the 12, 3, 6, and 9 o'clock positions.

12. When changing solutions during freeze substitution, it is important to remove as much liquid as possible without removing the sample. Within the solution chamber, aspirate from the bottom of the universal container which can be accessed through the hole in the scoring wheel spacer. This will draw the solution through the mesh bottom of the plastic vials preventing the loss of samples and remove most of the solution. The next solution can be added by dispensing the solution at the wall of the universal chamber. Do not fill the chamber above the top of the vials because samples can be washed over its walls and lost.

13. When moving a universal container filled with liquid nitrogen, insulated tweezers or pliers can be used to grasp the container. Once within the AFS chamber it is advisable that one hand is used to keep the container elevated, while the other moves the frozen sample into the freeze-substitution solution using insulated tweezers. Placing the liquid nitrogen-filled universal chamber on the floor of the AFS chamber will result in the boiling of the liquid nitrogen.

14. Do not panic when the acetone freezes as you place the frozen sample into the substitution solution. At -90°C, acetone is near its freezing temperature, the liquid nitrogen-cooled insulated tweezers used to transfer the frozen sample will often cool the acetone solution below its freezing temperature. Two strategies can be taken: (1) move the sample into acetone then retrieve the tweezers quickly before the acetone freezes. This strategy can work, however, you want to make sure that when you are moving your sample into acetone that it becomes submerged, in doing so the tweezers may become frozen in acetone. (2) Work slowly in the acetone to ensure the proper placement/submersion of the frozen sample into acetone, and then use another set of tweezers to scrape the acetone free from the frozen tweezers. This will require one to temporally place the liquid nitrogen-containing universal chamber in or near the AFS to free a hand for manipulating another set of tweezers. In the end, the acetone will quickly return to a liquid state with no detriment to the frozen sample.

15. This freeze-substitution protocol works well for visualizing membranes, in particular, plasma membranes, mitochondrial membranes, and synaptic vesicles, however, may over stain other structures, such as the contractile apparatus. Several other freeze-substitution protocols have been used to study the morphology of C. elegans (7,12,18,31) and may need to be considered when designing a freeze-substitution experiment.

16. At this point, the animals are encased in fixed E. coli. Although one could try to remove the bacteria and change the orientation of the animals, doing so introduces the possibility of damaging the fixed worms by mechanical forces before they are embedded.

17. If a needle is used to pry the sample free, first try scraping the inside wall of the freeze chamber to avoid damaging the animals within the bacteria.

18. Troubleshooting: several problems may arise during freezing and freeze substitution with tannic acid and osmium. These include undesirable dark staining of structures of interest (Fig. 2A, muscle m-line), ice crystal damage (Fig. 2C), poor resin infiltration (Fig. 2D), and poor resin polymerization. The first problem is specific for substitution with tannic acid and osmium: whereas membranes are well contrasted, other structures of interest may be too electron-opaque. As with most fixation techniques for electron microscopy, one condition will not be ideal for every study. To decrease staining, try decreasing the concentration of osmium and/or its incubation time at -25°C, or try staining without the use of tannic acid. Ice crystal damage may be introduced in several ways: improper freezing (consult the manufacturer's guidelines for testing the operation of the high-pressure freezer); the warming of the sample during its transfer to freeze substitution (try to work quickly to minimize the exposure of the sample to warmer environments); and the incomplete removal of water during freeze substitution (make sure the solutions used for substitution are anhydrous and that the low temperatures are maintained during substitution). Increase the duration of substitution at low temperatures if necessary. Resin infiltration can be problematic. The typical fix is to increase the duration during each step of infiltration. An additional problem can be the poor polymerization of resin, which hinders sectioning. Before diluting make sure to remove excess oxygen from the pure Araldite solution by applying a vacuum as oxygen can inhibit polymerization. If a block is poorly polymerized, try incubating it at 60°C for a longer duration.

19. This freeze-substitution protocol has proven successful for preserving immunore-activity of many protein types; however, because of the unpredictability of antigen preservation, it may not be ideal for every type of immunolabeling experiment. Other freeze-substitution protocols have been used to study the location of cellular proteins in C. elegans (11,17) and should be considered, as well as fixation conditions used for other sample types (31,33), when designing an experiment.

20. Similar to fixation with osmium, one may observe ice crystal damage and poor resin infiltration. If so, see Note 18. An additional problem specific to Lowicryl resins is its tendency to wick out of the mold, leaving the sample embedded in little or no resin. To prevent this, make sure the wall of the casting mold is not touching another surface and do not over fill the mold, but do not under fill as well. Because of the problematic nature of Lowicryl, it is advisable to try a mock infiltration/polymerization before starting your freeze-substitution experiment. Specifically, set up the AFS, and at low temperatures, try to polymerize a mold filled with resin.

21. Glycine washes are typically used to block unreacted aldehyde groups in sections containing tissues fixed in aldehydes, therefore, this step may be treated as optional if the above fixation protocol (Subheading 3.2.3.) is used.

22. The specificity of staining can be judged based on the signal-to-noise ratio where noise is defined as the density of gold particles when no primary antibody is used, when a preimmune sera is used as the primary antibody, within an area of the section that contains no worm tissue, or within a tissue of worm in which it is known the antigen is not expressed. A local significant increase in the density of gold particles above background signifies the distribution of the labeled protein. If background staining is high, repeat the staining experiment but vary blocking conditions, the concentration of primary antibody, and/or the concentration of secondary antibody. If background staining persists, try other postembedding immunostaining protocols (27,29,30), which may result in decreased background staining.

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