Optimized Fixation and Immunofluorescence Staining Methods for Dictyostelium Cells

Monica Hagedorn, Eva M. Neuhaus, and Thierry Soldati Summary

Recent years have seen a powerful revival of fluorescence microscopy techniques, both to observe live cells and fixed objects. The limits of sensitivity, simultaneous detection of multiple chromophores, and spatial resolution have all been pushed to the extreme. Therefore, it is essential to improve in parallel the quality of the structural and antigenic preservation during fixation and immunostaining. Chemical fixations are broadly used but often lead to antigenicity loss and severe membrane damages, such as organelle vesiculation. They also must be followed by membrane permeabilization by detergents or solvents, which can lead to extensive extraction and cytosol leakage. Fixation with solvents bypasses the need for permeabilization, but when carried out at "high" temperatures, leads to severe extraction of soluble proteins and lipids and cytosol wash-out, and has therefore been used routinely to visualize the cytoskeleton. Here, we describe a few modifications to the common aldehyde fixation protocol that help decrease the usual artifacts induced by chemical fixation. Alternatively, new techniques have now been established that are based on rapid freezing using a variety of coolants followed by fixation in solvents at low temperature. We present detailed protocols and notes that allow the achievement of optimal preservation and permeabilization for both light and electron microscopy.

Key Words: Immunofluorescence; microscopy; rapid freezing; fixation; detergents; preservation; structure; antigenicity; Dictyostelium.

1. Introduction

Recent years have seen a powerful revival of fluorescence microscopy techniques (1), both to observe live cells and fixed objects. The limits of sensitivity, simultaneous detection of multiple chromophores, and spatial resolution have all been pushed to the extreme (2). Nevertheless, very often, preparation of cells or tissues for immunocytochemistry, whether for light or electron microscopy

From: Methods in Molecular Biology, vol. 346: Dictyostelium discoideum Protocols Edited by: L. Eichinger and F. Rivero © Humana Press Inc., Totowa, NJ

(EM), takes its inspiration from cooking and witchcraft. All the recipes have in common the aim of solving the three major problems of preservation of antigenicity, preservation of native structure, and accessibility of the antigen. Despite about half a century of mostly empirical approaches, no optimal solution has been uncovered; instead, painstaking trial-and-error procedures are needed to determine the technique "best" suited to each particular case. It is not within the scope of this chapter to present an exhaustive theoretical and experimental list of the possible approaches and strategies, but the reader is advised to seek wisdom in such textbooks as the one from Gareth Griffiths (3) and further work from the authors. No panacea can be offered, but only a guide to the steps that can be optimized to reach the best compromise between the three parameters mentioned previously.

Briefly, in classical methods, fixation is responsible for the degree of preservation of both structure and antigenicity. Very often the improvement of one parameter is inversely proportional to the loss of the other: use of increased concentrations of aldehydes leads to better preservation of structure, but chemical modification of proteins (and other molecules) destroys the antibody binding sites. The same is generally true when switching from formaldehyde to glutaraldehyde, and most of the additives used in EM (osmium tetroxyde and other empirically selected chemicals) are deleterious to antibody recognition. Even when the optimal compromise between antigenicity and structure has been defined, accessibility remains a hurdle that is most often overcome by the use of detergents that either selectively or generically solubilize membrane lipids and create pores in the membranes. There is an empirical gradation from mild detergents (often specific to some lipids, e.g., sterols, such as digitonin and saponin) to stronger ones, such as Triton X100 or similar nonionic detergents. Depending on the strength of the fixation method used, detergents can remove or solubilize proteins and other small molecules, generating (sometimes desired) additional artifacts.

An alternative strategy has slowly emerged that bypasses the need for chemical denaturation of proteins and the use of detergents, namely the immobilization by freezing. Obviously, this approach has some drawbacks, mainly in the more sophisticated equipment needed, but offers serious advantages. Because the immobilization of all biological processes and biochemical machines happens almost instantaneously (at least one to two orders of magnitude faster compared with chemical fixations), there is little time for modification of the native structure. The major enemy is the formation and growth of ice crystals that leads to the appearance of artifactual hexagonal patterns inside the cell or organelles. Therefore, the ultimate goal of all the methods used—slamming against supercooled copper blocks, high-pressure freezing, and freezing on coverslips—is to generate amorphous ice. Whereas the first two methods can lead to several 100-^m thick, crystal-free samples, the rapid freezing on coverslips is limited to cell monolayers of up to 10-15 ^m thickness. The use of coolants such as liquid ethane has been shown to lead to excellent structure preservation when followed either by freeze substitution in solvents such as acetone or methanol or by resin embedding, but is rarely compatible with antigen detection. The most popular method for immuno-EM is likely the Tokuyashu method (4, and references therein), in which frozen sections are thawed and incubated with antibodies before visualzation, leading to remarkable antigenicity preservation; however, with this method, structure preservation is usually minimal. Recently, alternatives have been developed that take advantage of rapid cooling, either in liquid ethane for EM fine structure preservation or in methanol for light microscopy level preservation (5), and which are followed by fixation by coagulation or precipitation as a consequence of replacing the water by solvent around proteins and other molecules. This fixation results in excellent preservation of antigenicity, and precise temperature control can lead to graded levels of lipid extraction and thus of permeabili-zation, making the use of detergents unnecessary (5,6).

In summary, depending on the degree of structure and/or antigenicity preservation needed, the choice of the appropriate technique must be carefully tailored. Here, we present a robust, rapid-freezing, methanol fixation-permeabilization method and an alternative chemical fixation that is well adapted to Dictyostelium (an illustration of the results obtained can be found in Figs. 2 and 3, discussed later). It is also worth noting that optimization of the method is dependent on the structrure and the antigen that one uses to judge the improvements. We strongly recommend labeling with a contractile vacuole, as this is arguably the most complex and beautiful organelle in Dictyostelium. The preservation of its delicate structure, made of interconnected bladders and a reticular tubular network, is an excellent "standard meter" with which to monitor the efforts. Finally, use of 4',6-diamidino-2-phenylindole (DAPI) to stain nuclear DNA allows one to monitor the formation of hexagonal ice that might result from suboptimal freezing conditions inside the otherwise homogeneously stained nucleus.

2. Materials

2.1. Buffers, Fixatives, and Other Reagents

1. Soerensen buffer (SB): 15 mM KH2PO4, 2 mM Na2HPO4, pH 6.0.

2. Soerensen/Sorbitol buffer (SSB ): Soerensen buffer containing 120 mM sorbitol.

3. Phosphate-buffered saline (PBS): 140 mMNaCl, 2.7 mM KCl, 10 mMNa2HPO4, 1.8 mM KH2PO4.

4. Quenching buffer: PBS with 100 mM glycine

5. Blocking buffer: PBS with 0.2% gelatin and 0.1% Triton X100.

6. PIPES buffer: Piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) 20 mM buffered to pH 6.0 with NaOH.

7. Preparation of the saturated picric acid solution: dissolve 3 g of solid picric acid in 1 L of double-distilled (dd)-H2O and warm up to 80°C overnight. Leave to cool down to room temperature (crystals may build up and precipitate), set the pH to 6.0, and store at 4°C.

8. Preparation of the picric acid/paraformaldehyde fixative: always prepare fresh, in a 50 mL Falcon tube.

a. Weigh 0.4 g of paraformaldehyde (PAF) (powder, kept at 4°C). Caution should be exerted with PAF: do not inhale it, and always weigh it under a fume cabinet.

b. Add 10 mL 20 mM PIPES buffer.

c. Microwave in brief pulses until it dissolves (takes only a few seconds), then cool immediately (on ice) to room temperature.

d. Add 7 mL of dd-H2O (or accordingly less if you add 0.5-1.0 mL of 2.5 M sucrose in order to enhance preservation of filopodia).

e. Add 3 mL of a saturated picric acid solution (stock prepared in advance and kept at 4°C (see Subheading 2.1.1.).

2.2. Equipment

1. Coverslips: The choice of coverslip type is crucial. For conventional chemical fixation, standard coverslips (12 mm diameter, grade 1, approx 145 ^m thick, http://www.hecht-assistent.de) can be used, whereas for fixation in ultracold methanol, thinner glass coverslips (12 mm diameter, grade 0, approx 100 ^m thick) must be used. For rapid-freezing techniques using liquid ethane and destined to ultrastructural observations by EM, highest heat conductance is necessary, and thus 50-^m thick sapphire coverslips are used (3 or 6 mm diameter, Groh & Ripp, Germany, http://www.groh-ripp.de).

2. Liquid ethane manipulation: The ethane or propane can be directly condensed into a cold vessel (aluminium cup) set down on a small stage at the bottom of a styrofoam container filled with liquid nitrogen to the height of the stage. This should keep the environment around the liquid coolant purged of air. It may be necessary to refill the liquid nitrogen from time to time. If it happens that the coolant freezes, it can be thawed either by adding small volumes of liquid coolant or by touching the aluminium cup with a warm metal rod. Static electricity may be a real problem for liquid propane/ethane. Therefore, it is safer to use metal container and needles instead of glass or plastic (see also Chapter 21).

3. Freezing Dewar (FH Cryotec, Instrumentenbedarf Kryoelektronenmikroskopie, Plankstadt, Germany; schematically presented in Fig. 1): Alternatively, a styrofoam box with aluminium cups and racks can be used, similar to the one used in item 1. It should be placed in a -80°C freezer, taken out only briefly before plunging the coverslips (see Subheading 3.3.), and left at room tempera-

Grafix Biochemical

Fig. 1. Schematic drawing of the Dewar setup used for rapid freezing and ultracold-methanol fixation (see Subheading 3.3.). A metal chamber with an opening at the bottom is placed over a Dewar filled with liquid nitrogen. The temperature is controlled by varying the flux of liquid nitrogen vapor through the opening (A). The chamber is cooled to -85°C with the methanol-filled cups and racks (B) before the coverslips, with cells on top, are plunged at an angle of 15° (O) and transferred to the rack (C). Details of the coverslip rack are shown schematically in D. Next, the temperature is raised to -35°C in about 30 min (E) and the coverslips are transferred to PBS at room temperature (F). The methanol is diluted by moving the coverslip through the air-water interface (G).

Fig. 1. Schematic drawing of the Dewar setup used for rapid freezing and ultracold-methanol fixation (see Subheading 3.3.). A metal chamber with an opening at the bottom is placed over a Dewar filled with liquid nitrogen. The temperature is controlled by varying the flux of liquid nitrogen vapor through the opening (A). The chamber is cooled to -85°C with the methanol-filled cups and racks (B) before the coverslips, with cells on top, are plunged at an angle of 15° (O) and transferred to the rack (C). Details of the coverslip rack are shown schematically in D. Next, the temperature is raised to -35°C in about 30 min (E) and the coverslips are transferred to PBS at room temperature (F). The methanol is diluted by moving the coverslip through the air-water interface (G).

ture until the methanol reaches -35°C (see also Chapter 21).

4. Humid chamber: a humid chamber for immunostainings is easily made out of a large (20-cm diameter) Petri dish with a glass lid. The bottom is covered with clean Parafilm before each incubation, and paper towels soaked in water are pressed at the periphery. A dark bench coat facilitates visualization of coverslips, and a light-tight cover can be placed on top to prevent photobleaching.

3. Methods

A comparison of the results achieved using the different fixation procedures described are shown in Figs. 2 and 3.

3.1. Preparation of Coverslips and Cell Plating

Coverslips are usually cleaned as follows:

1. Place 100 glass coverslips (or fewer if expensive sapphire is used) in a glass beaker, with a glass dish as a lid to prevent splashing.

2. Immerse in a solution of 50% nitric acid in dd-H2O and incubate at room temperature for 2 h under a fume hood, swirling gently from time to time.

3. Decant the acid solution (can be re-used) and replace with an ample volume of dd-H2O to rinse beaker and coverslips; swirl gently for a few seconds and decant.

4. Repeat 5-10 times and follow with two similar rinses in pure ethanol.

5. Dry the coverslips in a microwave at full power until the coverslips are completely dry. Use a glass dish as a lid to prevent coverslips from "popping" out of the beaker.

6. Place the coverslips in a box, between layers of paper tissues. With this treatment, we usually do not need to sterilize the coverslips by autoclaving before plating the cells. Also, autoclaving sometimes induces coverslip bending.

7. Place up to eight coverslips at the bottom of a 6-cm diameter plastic dish, and plate cells at an adequate density so as to reach 70-80% confluency after overnight growth.

8. Before fixation, cells can be treated as necessary, for example by feeding fluid phase markers or particles.

3.2. Rapid Freezing in Liquid Ethane

This step is necessary for achieving the highest degree of structure preservation, and is useful for observation of delicate and/or transient structures. This is

Fig. 2. (opposite page) Effects of fixation on structure preservation. Fluorescence microscopy of Dictyostelium discoideum cells stained against the vacuolar H+-AT-Pase in order to visualize the contractile vacuole system. Each row of images represents three maximum intensity projections of equal number of optical sections through one cell. The total number of sections for A-C was 21; for D-F and G-I it was 18. Stack pictures were taken using a Leica, AS MDW-widefield microscope equipped with a 100x objective on a piezo z-positioner and a charge-coupled device camera. Raw o c re

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