CYTOSKELETON

This material was originally published in the Purdue Cytometry CD-ROM Series,volume 4

CYTOSKELETON Carla Fiorentini*, Paola Matarrese, Alessia Fabbri and Walter Malorni Department of Ultrastructures, Istituto Superiore di Sanità, Viale Regina Elena 299, OO161 Rome, Italy *Corresponding author: fax: +39-6-49387140; email: fiorentini@ul.net.iss.it

Introduction

This chapter is dedicated to the principal techniques to analyze the cytoskeleton, the most appropriate being the immunocytochemical methods which will be herein detailed. Other techniques, such as DNA transfection and microinjection, which also offer a valid contribution to the analysis of the cytoskeleton in fluorescence microscopy, will be only briefly introduced in this chapter. For further information, we suggest to consider ref. 2 (for DNA transfection) and ref. 3 (for microinjection).

DNA transfection follows the cloning of a gene, reintroducing it into various eukaryotic cell lines and thus allowing the study of the protein-encoded characteristics. In fact, by using different techniques, cells can be induced to internalize the gene of interest expressed by eukaryotic vector. Both normal or manipulated genes can be transfected in cells and, by this way, transfection represent a very useful method to elucidate structure-function relationships. Microinjection allows access of various molecules to cells like the ones to which cells are impermeable, antibodies for intracellular proteins and cellular proteins fused to fluorescent markers. Whatever the way used to introduce such molecules (glass micropipette, vesicle etc.), this technique offers the possibility of studying the function of a protein as well as the dynamic of some structures.

However, as stated above, the elective experimental methods for studying the cytoskeleton during apoptosis are immunocytochemical techniques (4), such as fluorescence microscopy, flow cytometry and immunoprecipitation (5). They will offer useful approaches to study qualitative and quantitative alterations of the cell cytoskeleton as well as of a number of proteins regulating the cytoskeleton state, also in response to an apoptotic stimulus.

This chapter can be useful considering that the field we are dealing with has developed so rapidly in the last few years that a novice may encounter difficulty selecting an appropriate technique, performing the various steps of the procedure correctly, and interpreting results. The following three techniques will be detailed:

A) Fluorescence microscopy;

B) Flow cytometry;

C) Immunoprecipitation.

Fluorescence microscopy

A.1 Introduction

The fluorescence-labelled antibody method developed by Coons and Kaplan in 1950 (6) was one of the first immunocytochemical techniques described and it has been used extensively over the last two decades. The method can be either direct or indirect and is based on the fact that once a fluorocrome-labelled antibody bound to the specific antigen is exposed to visible or ultraviolet light, a fluorescent light (characteristic for each different fluorocrome) will appear at the site of antigen-antibody binding. This fluorescence can be observed with the light microscope, allowing the determination of the antigen presence or absence. A.2 Protocol

In this protocol section we will consider only isolated cells, either adherent on a substrate or floating in the medium. A similar procedure can also be adopted for tissues. A.2.1 Materials

A.2.2 Methods

Standard protocol

Perform steps from 1 to 3 only for floating cells. For cells grown on coverslips consider step 4 as the first.

Centrifuge cells at 600 rpm for 5 min.
Resuspend the pellet in 40 µl of PBS (see Appendix A1).
Deposit 30 µl of cell suspension on a coverslips coated with poly-L-lysine (as reported in Appendix 1) for 20 min.
Rinse rapidly the cells adhering to the coverslip with medium devoid of serum.
Fix cells with paraformaldehyde solution (see Appendix 1) for 10 min.
Wash cells 3 times with PBS buffer.
Permeabilize cells with Triton X-100 (see Appendix A1) for 5 min.
Preincubate cells with PBS 1% heat-inactivated FCS or BSA, for 15 min to prevent unspecific immunoglobulin adsorption.
Incubate the cells with specific antibody for 30 min at 37°C.
Wash cells 3 times with PBS buffer.
For indirect method, incubate the cells with labelled second antibody for 30 min at room temperature. For direct method go directly to step 13.
Wash cells 3 times with PBS buffer.
Mount the coverslips with glycerol-PBS (2:1).
Analyze with a fluorescence microscope.

Protocol advised for cytokeratins' detection

Incubate cells in methyl alcohol for 10 min at 4°C
Dip coverslips for 20 seconds in acetone at -20°C.
Go to step 8 of the standard protocol.

Protocol advised for polymerized actin (F-actin) detection

F-actin can be detected either by a specific antibody against actin or by phalloidin (a well-known toxin which specifically binds F-actin) labelled to a fluorocrome (Figures 1 and 2). In the second case, we can adopt the same protocol as for a direct immunofluorescence. Step 8 of the standard protocol is not required. Protocol advised for antigens easily degradable

A3. Commentary

A3.1. Background Information

Immunofluorescence technique can be divided into two different methods of staining: i) a direct one involving the use of a primary antibody conjugated to the label and consisting in a one-step procedure; ii) an indirect staining that employs a primary antibody followed by a labelled secondary antibody. The choice between direct or indirect experimental procedure has to be made by the operator on the bases of: i) commercial findly of the antibody against the proteins of his interest; ii) quantity of antigen expressed by the cell. In fact, the indirect technique is able to amplify the fluorescence signal making this technique particularly indicated for detection of scarcely expressed antigens. A3.2. Critical Parameters 1. The affinity of the antibody for the antigen. Polyclonal or monoclonal antibodies can be used for detection of cytoskeletal proteins, although the use of polyclonal antibodies also causes higher non-specific backgrounds.

2. The choice of an appropriate experimental protocol for detecting the cytoskeletal proteins of interest.

A3.3. Troubleshooting

Other problems that we can encounter by using this technique are the following:

False-negative results

- Modification or loss of the antigenic sites during fixation. In this case can be useful to modify the fixative solution or the kind of fixative used.

- Lack of antibody penetration for incomplete permeabilization. It is possible bypass this problem prolonging the time of step 7 of the standard protocol or to use a more concentrated solution of permeabilizing agent.

- Sterical hindrance or the masking of antigenic sites by proteins. In this particular case it can be useful to use, after cell fixation, a detergent solution for a few minutes. Wash and then go on as indicated in the standard protocol.

False-positive results

- Presence of free reactive groups after aldehyde fixation. These groups can be still present into the cells leading to non-immunological binding of proteins (e.g. IgGs). This can be prevented by incubation of the cells with serum not recognized by the immunoreagents or by the use of buffers containing small molecules (lysine, glycine, tris) or proteins (gelatine, serum albumin), both with reactive amino groups.

- Hydrophobic and/or ionic interactions. Ionic interactions of antibodies (Fc region) with cellular components such as nuclear and ribosomal components, can be minimized by the use of buffer with enhanced ionic strength. Hydrophobic interactions can result from hydrophobic parts present in antibodies and labels with hydrophobic cellular compounds. The addition of detergents in washings fluids and incubation media might reduce this interaction. Moreover, to minimize the influence of ionic charges, it was found that incubating the tissue section at pH 8.6 results in a decrease in background levels. The rationale behind this treatment is that IgGs have an isoelectric point of 8.6, which means that incubation at this pH results in a minimal charge on the IgG molecules.

- Autofluorescence of cellular components such as flavonoids etc.

- Although nonspecific staining may be avoid with precautionary measures, each study dealing with a particular antigen should include a series of controls in order to ensure that non specific localization occurred. The most obvious control is one that uses normal serum in place of, and from the same animal source as, the primary antibody. While this does not preclude the possibility that the primary antibody is staining something other than the antigen being sought, it does give some indication of back-ground or nonspecific staining due to other components in the tissue or components in the reagents being used. In addition to normal serum, both negative and positive control tissue should be used in order to detect discrepancies from one experiment to the next. The use of antibodies directed against irrelevant antigens is also helpful in determining whether the animal used for antibody production contains naturally occurring, or nonspecific, antibodies against irrelevant antigens other than the one under study. In such cases, localization would be expected to occur regardless of primary antibody specificity. The absorption of primary antibody along with its antigen is perhaps the most definitive control, but this occurrence is not an absolute guarantee that the correct antigen has been localized. This lack of certainty can best be explained by examining the binding of monoclonal antibodies. Such antibodies are against a very small portion of the protein, amounting to approximately seven amino acids. It is not unreasonable to imagine that more than one protein in a given tissue may contain a similar sequence of seven amino-acids. Still, the use of appropriately purified reagents and sufficient controls can greatly reduce the opportunity for error.

A3.4. Anticipated Results

Recent works evidenziate the involvement of the cell cytoskeleton in the apoptotic process (1). It can therefore be useful to evaluate the qualitative alterations of cytoskeletal components such as tubulin, actin, actin binding proteins, etc. which accompany cell death. It is fundamental, however, to distinguish between real alterations of the cytoskeleton and artefacts due to inappropriate experimental procedures. A3.5. Time Considerations

The immunofluorescence technique is an easy and very rapid technique which requires less than one day of work to be performed. Appendix A1

Phosphate Buffer Solution (PBS) 1x

₄

₂

₄

₂

Paraformaldehyde fixative solution

Triton permeating solution

Saponine permeating solution

Nonidet permeating solution

Poly-L-lysine solution

₂

For preparation of coated coverslips: deposit 40 m l of this solution on 13 mm coverslips and wait the complete evaporation. Store the coverslips at room temperature protecting them from the dust.

Appendix A2

Appendix A3

B) Flow cytometry B1. Introduction

The quantification of cytoskeletal proteins can be obtained by using flow cytometry. Measurements of fluorescent dyes bound to cellular constituents represent the majority of flow cytometric applications. Fluorescence has three major advantages in flow studies: i) fluorescence emission is direcltly proportional to specific cell constituents; ii) low concentration of dye per cell can be detected; and iii) nonfluorescent compounds can be made fluorescent by intracellular enzymes.

B2. Protocol

B2.1. Materials

B2.2. Methods

Standard protocol for cytoskeletal proteins' detection by flow cytometry

Harvest the cells with 10 mM EDTA and trypsin solutions.

Wash the cell suspension with PBS
Centrifuge cells at 800 rpm for 5 min.
Resuspend 10⁶ cells in 1 ml of PBS (see Appendix B1).
Add 2 ml of paraformaldehyde solution (see Appendix B1) for 10 min at room temperature.
Wash cells twice with PBS buffer.
Centrifuge cells at 800 rpm for 5 min.
Resuspend cells in 1 ml of cold PBS.
Add 1 ml of cold (-20°C) methyl alcohol
Incubate 10 min at 4°C.
Wash cells twice with PBS buffer.
Resuspend cells in 50 m l PBS-Nonidet P-40 0.5 % (see Appendix B1).
Incubate cells with specific antibody for 1 hour at room temperature.
Wash cells twice with PBS buffer.
For the indirect method, incubate the cells with labelled second antibody for 30 min at room temperature. For the direct method go directly to step 15.
Wash cells twice with PBS buffer.
Resuspend cells in 500 µl of cold PBS.
Analyze with a cytofluorimeter.

Protocol advised for antigens easily degradable

Repeat steps from 1 to 4 of the standard protocol.

5. Add 2 ml of paraformaldehyde solution (see Appendix B1) for 10 min at 4°C.

6. Add 200 m l of Triton solution (see Appendix B1) for 10 min at 4°C.

7. Wash cells twice with cold PBS buffer.

8. Centrifuge cells at 800 rpm for 5 min.

9. Resuspend cells in 1 ml of 1% AB human serum in cold PBS.

10. Wash cells with PBS buffer.

11. Resuspend cells in 50 m l of 1% AB human serum in cold (see Appendix B1).

12. Incubate cells with specific antibody for 1 hour at 4°C.

13. Wash cells twice with PBS buffer.

14. For the indirect method, incubate the cells with labelled second antibody for 30 min at 4°C. For the direct method go directly to step 16.

15. Wash cells twice with PBS.

16. Resuspend cells in 500 m l of cold PBS.

17. Analyze with a cytofluorimeter.

B3. Commentary

B3.1. Background Information
See A3.1

B3.2. Critical Parameters
See A3.2.

B3.3. Troubleshooting

Although presenting most of the disavantages of fluorescence microscopy (see A.3.3.), flow cytometry has the advantages of: i) a major sensitivity of the cytometer with respect to the fluorescence microscope; ii) a higher rapidity in acquiring and recording data. B3.4. Anticipated Results

The quantitative alterations of cytoskeletal components, such as for example polimerized actin, can in certain cases be associated with cell death hinderance (10). It is fundamental, however, to distinguish between real changes in quantity of the cytoskeletal proteins and artefacts due to inappropriate experimental procedures. For this reason is very important to adjust the cytometer by using the specific negative controls.

All problems relative to flow cytometry tecnique are discussed in detail in the chapters of this book specific for flow cytometry.

B3.5. Time Considerations

The flow cytometry technique is a very rapid technique which require a day of work to be performed. Appendix B1

Phosphate Buffer Solution (PBS) 1x

KCl 0.25 g

NaHPO₄ x 2 H₂O 1.44 g

KH2PO₄ 0.25 g

Add 1 L of distilled H₂O.

pH from 7.2 to 7.4.

Store at 4°C for a few weeks.

EDTA solution

in PBS pH 7.4.

Store at 4°C for a few months.

Trypsin solution

in PBS pH 7.4

Store at 4°C for a few months.

Paraformaldehyde fixative solution

in PBS pH 7.4.

Store at 4°C in the dark for a few days.

Triton permeating solution

in PBS pH 7.4.

Store at room temperature for a few weeks.

Nonidet permeating solution

in PBS pH 7.4.

Store at 4°C for few days.

Appendix B2

3. Methyl alcohol Carlo Erba (cat. n. 414816)

4. Nonidet P-40 Sigma (cat. n. N-6507)

5. Paraformaldehyde ICN (cat. n. 17538)

6. Triton X-100 Sigma (cat. n. 9002-93-1)

7. Trypsin ICN (cat. n. 101179)

Appendix B3

2. Cytometer, equipped with a computer.

C) Immunoprecipitation

C1. Introduction

Recent advances in such a field combined DNA transfection with immunoprecipitation, thus offering a contribution to the analysis of proteins in general and of proteins of the cytoskeleton in particular (see for example ref 7). A clear improvement of the coupling of these two techniques is the fact that not all proteins have antibodies capable of immunoprecipitating, thus it is possible to manipulate the gene introduced by transfection so that it can bring an artificial epitope. For example, one of the most useful epitope utilized is the protein G of VSV, recognized by the monoclonal antibody P5D4. By introducing a protein tagged by this epitope, the protein itself acquire the capacity of being immunoprecipitated, thus rendering easier the study of the protein characteristics.

C2. Protocol

- lysis of the cells to release the antigen and preclearing of lysates

- formation of the antibody-antigen complex

- purification of the immune complex

C2.1. Materials

2. NaCl

3. Trizma base

4. Protein A/G Agarose

5. Leammli buffer

C2.2. Methods

Lysis of cells and preclearing lysate

1. Wash cells with PBS at room temperature. Drain well.

2. Incubate cells in lysis buffer A (10⁷ cells in 1 ml buffer prechilled to 4°C) for 1 h at 4°C.

3. Scrape the cells and debris from the plate with a rubber policeman and transfer all the preparation to a 1.5 ml conical tube.

4. Centrifuge 10 min at 10,000 g at 4°C.

5. (Optional) Centrifuge the supernatant at 100,000 g for 30 min.

6. Quantify the protein concentration and use the same amount of proteins for all samples.

7. Preclear supernatant by adding 20 m l of control Protein A/G agarose prepared without antibody or coupled to non specific antibody.

8. Shake on an orbital shaker for 1 h at 4°C.

9. Centrifuge 1 min at 10,000 g and save the supernatant.

(Optional from 7 to 9)

Forming the antibody-antigen complex

11. Incubate 1 h on ice

Purification of the immune complex 12. Add 20 m l of Protein A/G plus Agarose and incubate on an orbital shaker at 4°C for 1 h.

13. Centrifuge at 10,000 g for 1 min at 4°C.

14. Remove the supernatant by aspiration and add 0.5 ml of lysis buffer. Vortex to resuspend.

15. Wash three times with lysis buffer. Remove the last wash as completely as possible.

16. Add 20 m l of Leammli sample buffer 1X. Heat for 5 min at 100°C, centrifuge at full speed and use the released immune complex for SDS-polyacrilamide gel electrophoresis (SDS-PAGE) (5).

17. Detection of the immunoprecipitation reaction can be performed with the following methods:

a) Comassie Brilliant Blue or Silver staining (ref 5) if the amount of protein is >1 m g.

b) Transfer to nitrocellulose and blotting (ref 5) if the amount of proteins is <1 m g.

c) Autoradiography (ref 6) if you labelled with radioactive amino acid to detect very rare proteins

C3. Commentary

C3.1. Background information

In itself, this technique is a very simple one, but it takes time and accuracy to find the right working conditions, almost every antigen requires a particular protocol "treatment".

C3.2. Critical parameters 1. As mentioned above, the affinity of the antibody for the antigen is one of the critical parameters for good resolution in immunoprecipitation. This gives the strength of the binding of an epitope to an antibody and since immunoprecipitation relies on the formation of antigen-antibody complex in solution at relatively low concentration of the antigen, affinity of at least 108 mol-1 are required. Polyclonal antibodies are the most commonly used for immunoprecipitation. They contain antibody that bind to multiple sites on the antigen, thus providing a high affinity for the antigen and a more stable antigen-antibody-protein A complex. Although using polyclonal antibodies for immunoprecipitation often produce stable multivalent interactions, their use also causes higher non-specific backgrounds. Monoclonal antibodies bind to only one epitope thus providing a specific tool to identify a particular structure on an antigen. In addition, the immune complexes formed using monoclonal antibodies give less of a problem with non-specific binding, the backgrounds, in general, being cleaner. Although monoclonal antibodies may solve problems of background, their use also creates some difficulties. The major problem is given by the affinity. Because the antigen is held, in general, by one antibody-antigen interaction, monoclonal antibodies with affinities lower than 107 mol-1 are difficult to use in immunoprecipitation. Another problem by using monoclonal antibodies is the possibility of detecting cross-reactions with other polypeptides. An epitope, in fact, can be a relatively small structure (5-7 aminoacids), thus there is a reasonable chance that a similar epitope can be found on another polypeptide.

2. In general, the conditions used for lysis should be as gentle as possible to retain the antibody binding site and to avoid solubilizing background proteins, but they have to ensure quantitative release of the antigen. Salt concentration, type of detergent, presence of divalent cations and pH are all variables that affect the release of polypeptide antigens. The following parameters should be monitored to determine the optimal conditions of extraction:

- salt concentrations between 0 and 1 M

- non ionic detergent concentrations between 0.1 and 2%

- ionic detergent concentrations between 0.01 and 0.5%

- divalent cations concentrations between 0 and 10 mM

- pH between 6 and 9

3. A gel run after immunoprecipitation contains not only the protein of interest, but also the antibodies as one of the major band. Antibodies are formed by light and heavy chain which together have a molecular weight of about 150 kDa. The light chain alone is at the MW of many important proteins which are usually studied by immunoprecipitation. Thus, in the case of Comassie blue, silver staining and immunoblotting is recommended to use a Laemmli buffer without DTT so that the two chains are not separated during migration and they can be found as a unique band at almost 150 kDa. Also, to avoid this problem in immunoblotting, different antibodies might be used to immunoprecipitate and to detect the protein after transfer, i.e. polyclonal antibodies to immunoprecipitate and monoclonal antibodies to detect the protein. Thus, the secondary antibody will be an anti-mouse which will not recognize the polyclonal immunoglobulin used to immunoprecipitate.

C3.3. Troubleshooting

The most frequent problem is the presence of non-specific background which may be reduced by the following recommendations:

1. Perform the preclearing step, reported in the text as optional.

2. Add proteins such as BSA, gelatine to the lysate

3. Preincubate protein A/G Agarose with 2% BSA prior adding to lysate

4. Use more stringent washes such as 1 M NaCl.

5. Increase the number of wash cycles

6. Try a different antibody

C3.4. Anticipated results

Various works have been published so far in which both types of proteins have been immunoprecipitated. In particular see ref. 8.

C3.5. Time considerations

The immunoprecipitation, in itself, is an easy technique that in a day can be performed. Appendix C1

Phosphate Buffer Solution (PBS) 1x

KCl 0.25 g

NaHPO₄ x 2 H₂O 1.44 g

KH2PO₄ 0.25 g

Add 1 L of distilled H₂O.

pH from 7.2 to 7.4.

Store at 4°C for a few weeks.

Lysis buffer A

Na Cl 150 mM

Triton X-100 0.5% (see also critical parameters)

Store at 4°C for a few weeks

Leammli buffer 1(x)

Trizma base 60 mM

glycerol 10%

DTT 100 mM

bromophenol bleu 0.1%

Store at -20°C for months

Comassie Brilliant Blue

Methanol:H₂O (1:1 v/v) 90 ml

glacial acetic acid 10 ml

Filter the solution before use.

Store at room temperature for a few months

Appendix C2

2. Protein A/G plus Agarose Santa Cruz Biotechnology (cat. n. sc 2003)

3. Glacial acetic acid Prolabo (cat. n. 20103.320)

4. Comassie brilliant blue Sigma (cat. n. B-0149)

Appendix C3

1. Ultracentrifuge

Key References

1. Meredith, J.E. Jr, and M.A. Schwartz. 1997. Integrins, adhesion and apoptosis. Trends Cell Biol. 7, 146-150.

2. Sambrook, J., E.F. Fritsch, T. Maniatis. 1989. Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, eds.

3. Ridley, A.J., and A. Hall. 1992. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70: 389-399.

4. Immunocytochemical technology. G. V. Childs, ed. Alan R. Liss, Inc., New York.

5. Harlow, E., D. Lane. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, eds.

6. Coons, A.H., and M.H. Kaplan. 1950. Localization of antigens in tissue cells. Improvements in a method for the detection of antigen by means of fluorescent antibody. Journal Experimental Medicine 91: 1-13.

7. Wary, K.K, F. Maniero, S.J. Isakoff, E.E. Marcantonio, and F.G. Giancotti. 1996. The adaptor protein shc couples a class of integrins to the control of cell cycle progression. Cell 87 (4): 733-742.

8. Defilippi, P., M. Venturino, D. Gulino, A. Duperray, P. Boquet, C. Fiorentini, G. Volpe, M. Palmieri, L. Silengo, and G. Tarone. 1997. Dissection of pathways implicated in integrin-mediated actin cytoskeleton assembly. J. Biol. Chem. 272: 21726-21734.

9. Rouslahti, E. 1997. Stretching is good for a cell. Science 276, 1345-1346.

10. Fiorentini, C., A. Fabbri, P. Matarrese, L. Falzano, P. Boquet, and W. Malorni. 1997. Hinderance of apoptosis and phagocytic behaviour induced by E. coli cytotoxic necrotizing factor 1 (CNF1): two related activities in epithelial cells. Biophys. Biochem. Res. Com. 241:341-346.

11. Haldar, S., A. Basu, and C.M. Croce. 1997. Bcl2 is the guardian of microtubule integrity. Cancer Res. 15: 229-233.

FIGURE LEGENDS

Figure 1.

Fluorescence microscopy of human epithelial cells stained for the detection of F-actin (A) and of the actin-binding protein alpha-actinin (B). Following the procedure reported in Methods A2.2., F-actin was directly evidenced with FITC-phalloidin whereas alpha-actinin by an indirect method, standard protocol, using a specific monoclonal antibody (Chemicon). To note F-actin organized in stress fibers and alpha-actinin distributed along the actin filaments.

Figure 2.

Fluorescence microscopy of human epithelial cells stained with FITC-phalloidin for F-actin detection, as reported in Methods A.2.2.. The figure shows some of the actin cytoskeletal alterations which have been associated with apoptosis. Spreading cells, characterized by membrane ruffling (A) and by a peculiar thin meshwork (B) are known to protect cells against apoptosis (9). By contrast, apoptosis often causes the rounding up and the breakdown of the actin network (10).

Figure 3.

Fluorescence microscopy of human epithelial cells stained for microtubular network (A) and Bcl-XL detection (B). Bcl-XL is an anti-apoptotic protein of the Bcl-2 family, associated with microtubules (11). In both cases, it was used the indirect method reported in the standard protocol, Methods A.2.2. Tubulin was evidenced by using a mixture (1/1) of monoclonal anti a and b tubulin (Sigma) and Bcl-XL with a specific anti rabbit polyclonal antibody (Santa Cruz). To note the distribution of the anti-apoptotic protein which overlaps the microtubular network.

Figure 4.

Fluorescence microscopy of human epithelial cells stained for the detection of the intermediate filament-type vimentin. Vimentin was evidenced by a monoclonal antibody (Sigma) following the indirect method described in Methods A.2.2. To note the arrangement of vimentin in control cells (A) and the breakdown of its network (B) upon exposure to an apoptotic inducer (in the micrograph is shown the effect of a bacterial toxin known to cause apoptosis in epithelial cells).

Figure 5.

Fluorescence microscopy of human epithelial cells stained for cytokeratins' detection. A monoclonal antibody anti-pan-cytokeratin (Sigma) was adopted in the indirect method described in Methods A.2.2. To note that the cytokeratin network in epithelial cells (A) can undergo collapse when treated with apoptotic agents which influence the cell shape (B).