ARTICLE |
Correspondence to: Mark C. Willingham, Dept. of Pathology, Wake Forest University School of Medicine, WinstonSalem, NC 271571072. E-mail: mwilling@wfubmc.edu
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Apoptosis has gained central importance in the study of many biological processes, including neoplasia, neurodegenerative diseases, and development. One of the limitations of many studies is the difficulty of specifically identifying individual apoptotic cells. Of the many specific methods developed to detect apoptotic cells, most are not applicable to histological sections of archival paraffin-embedded tissues. Recently, advances in the understanding of the molecular events in apoptosis have led to the realization that caspase activation is by far the most specific indicator of this cell suicide mechanism. Several publications have reported the development of antibodies directed at neoepitopes that are generated in various substrates through the action of caspases. One of these is that present on activated caspase 3, a ubiquitously distributed caspase that is a main effector caspase of the apoptotic cascade within cells. This study demonstrates the utility of using a recently commercially available antibody to cleaved caspase 3 in archival paraffin sections, suggesting that this may be a highly specific and sensitive method generally applicable to many studies of archival material. (J Histochem Cytochem 50:449454, 2002)
Key Words: apoptosis, caspase, immunohistochemistry, TNF, ricin
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
APOPTOSIS is a highly regulated cell suicide mechanism that is important for many biological processes, including embryonic development, response of tumors to cancer chemotherapy, and the pathogenesis of neurodegenerative diseases (
Because archival material represents a potential wealth of information that could be employed to study many disease processes, there has been considerable interest in developing methods that could specifically demonstrate apoptotic cells in paraffin sections. Through the study of the molecular events in apoptosis systems, it is now clear that cleavage of protein substrates by caspases is a pivotal cascade that is unique to apoptotic cells (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture and Reagents
KB human carcinoma cells were obtained from American Type Culture Collection (Rockville, MD) and propagated in RPMI-1640 medium supplemented with 10% fetal calf serum, penicillin, and streptomycin at 37C in a 5% CO2/95% air atmosphere. Ricin, TNF, cycloheximide, and culture media were obtained from Sigma Chemicals (St Louis, MO). Rabbit polyclonal antibody to cleaved caspase 3 was obtained from Cell Signaling Technologies (Beverly, MA; catalog #9661). Affinity-purified goat anti-rabbit IgG labeled with rhodamine was obtained from Jackson ImmunoResearch (West Grove, PA).
Tissues
Blocks of paraffin-embedded tissues that had been formalin-fixed were obtained from the archives of PhenoPath Laboratories, corresponding to normal human tissues, including lung, liver, breast, lymph node, skin, brain, kidney, pancreas, intestine, and skeletal muscle.
Apoptosis Experiments
For long-term apoptosis experiments, KB cells in T-75 flasks were treated with either ricin (1 ng/ml) or a mixture of tumor necrosis factor (TNF; 10 ng/ml) together with cycloheximide (5 µg/ml) for 2 days. The floating cells from treated flasks were then harvested at room temperature (RT) using a Shandon Cytospin centrifuge, fixed in 3.7% formaldehyde for 15 min, and permeabilized using 0.1% Triton X-100 for 5 min. The cytospins were then incubated in rabbit anti-cleaved caspase 3 antibody (1:100 dilution of manufacturer's stock) in 1% bovine serum albumin with PBS for 30 min. After a PBS wash, the cells were incubated with 25 µg/ml affinity-purified goat anti-rabbit IgG conjugated with rhodamine for 30 min. After washing and postfixation in formaldehyde, the preparations were incubated with 0.1 µg/ml DAPI in methanol and mounted under a coverslip in glycerol.
For shorter-term apoptosis experiments, two T-75 flasks of KB cells were incubated with 1 µg/ml ricin for 7 hr, and the floating cells were removed by shaking and pelleted by centrifugation at 5000 x g in 1% BSAPBS. Similarly, untreated control cells were removed from flasks by trypsinization and pelleted in 1% BSAPBS. Small aliquots of these preparations before centrifugation were also used to prepare cytopreps that were fixed in acetone and stained with hematoxylin. The pellets were fixed by slowly dripping 3.7% formaldehyde in PBS on top of the solid pellets and incubating overnight at RT. The pellets were then gently removed from the centrifuge tubes, placed in filter paper and into a cassette, and routinely processed for paraffin embedding.
Immunohistochemistry using the rabbit anti-cleaved caspase 3 antibody was performed on deparaffinized tissue sections using a routine avidinbiotinimmunoperoxidase technique [Vectastain Elite avidinbiotinimmunoperoxidase kit reagents (Vector Labs, Burlingame, CA)]. Before incubation with the primary antibody (1:200 dilution as supplied by the manufacturer), tissue sections were subjected to heat-induced epitope retrieval by incubation in a pH 8.0 0.01 M EDTA solution for 10 min in a vegetable steamer, followed by 20-min cool-down and treatment with 3% hydrogen peroxide before antibody application.
Microscopy
Fluorescence images were prepared using a Zeiss Axioplan 2 microscope equipped with phase-contrast optics and with rhodamine and UV channel filters. Images were recorded using either a Dage 300 monochrome CCD camera or a Zeiss Axiocam CCD camera, and composite figures were prepared using Adobe Photoshop.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A polyclonal affinity-purified rabbit antibody to the neoepitope peptide at the end of cleaved caspase 3 (1720-kD fragments) was obtained from Cell Signaling Technologies (catalog #9661). This antibody reacts with this neoepitope in mouse, rat, and human caspase 3. To evaluate its utility in human cells, we employed a model apoptosis sytem in KB human carcinoma cells that we have previously described, using either TNF or ricin to induce apoptosis. Cells in culture exposed to either of these agents for 2 days were then fixed in formaldehyde, permeabilized with Triton X-100, and reacted with rabbit anti-cleaved caspase 3, followed by anti-rabbit IgG conjugated to rhodamine. These cells were further incubated with DAPI to visualize nuclear DNA. Fig 1 demonstrates the appearance of apoptotic and normal cells using this method. Cells with normal nuclear morphology showed no detectable reaction with this antibody, but cells with apoptotic nuclear and surface morphology showed various amounts of reactivity in a diffuse or punctate cytoplasmic pattern. The strength of this signal suggested that there was sufficient caspase 3 in such cells to allow easy detection of its catalysis during apoptosis.
|
To determine if this signal would be detectable after fixation and routine processing into paraffin, cell pellets were prepared from such cells treated with 1 µg/ml ricin for 7 hr in culture. These pellets were then fixed in formaldehyde, dehydrated through ethanol and xylene, and embedded in paraffin by routine methods. Sections from these blocks were then subjected to immunohistochemistry using anti-cleaved caspase 3 and labeling with anti-rabbit IgG conjugated to horseradish peroxidase, followed by development using diaminobenzidine. Fig 2 shows images from these sections, which clearly display a strong signal in the cytoplasm of such apoptotic cells and no such reaction in cells with normal morphology. Therefore, this antibody could clearly specifically distinguish cells in apoptosis from normal cells.
|
To further evaluate the utility of this approach, we employed a variety of archival blocks of normal human tissues. Most tissues showed no significant immunostaining, although prominent immunostaining was noted corresponding to the apoptotic cells present within the germinal centers of normal lymph nodes. In many tissues, cytoplasmic signal was also noted in polymorphonuclear leukocytes. Apoptosis of neutrophils has been previously described (reviewed in
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We and others have previously presented reviews of the various methods available for detection of apoptotic cells (
It is now clear that caspase activation (cleavage of procaspase to active caspase) is a hallmark of almost all apoptotic systems (
Not all such antibodies may work well in archival material because of the nature of the peptide or the levels of the specific caspase or other substrate present in cells. The neoepitope of cleaved caspase 3 should, however, be fairly ubiquitous and of sufficient levels to be useful in most cell types. The crossreactivity of this antibody with this neoepitope in major mammalian species used clinically and in experimental studies makes this antibody very useful. Evaluation of this and other caspase cleavage epitopes should provide a generally useful tool to identify apoptotic cells in archival material, expanding the range of experimental and disease settings in which apoptosis can be accurately evaluated.
Received for publication December 7, 2001; accepted December 12, 2001.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barrett KL, Willingham J, Garvin AJ, Willingham MC (2001) Advances in cytochemical methods for detection of apoptosis. J Histochem Cytochem 49:821-832
Caulin C, Salveson GS, Oshima RG (1997) Caspase cleavage of cytokeratin 18 and reorganization of intermediate filaments during epithelial apoptosis. J Cell Biol 138:1379-1394
Collins JA, Schandl CA, Young KK, Vesely J, Willingham MC (1997) Major DNA fragmentation is a late event in apoptosis. J Histochem Cytochem 45:923-934
deBoer RA, van Veldhuisen DJ, van der Wijk J, Brouwer RM, de Jonge N, Cole GM, Suurmeijer AJ (2000) Additional use of immunostaining for active caspase 3 and cleaved actin and PARP fragments to detect apoptosis in patients with chronic heart failure. J Cardiac Failure 6:330-337[Medline]
Green DR (2000) Apoptotic pathways: paper wraps stone blunts scissors. Cell 102:1-4[Medline]
Huppertz B, Frank H-G, Kaufmann P (1999) The apoptosis cascademorphological and immunohistochemical methods for its visualization. Anat Embryol 200:1-18[Medline]
Knaapen M, DeBie M, Muhring J, Kockx M (1999) Cleaved PARP as a marker for apoptosis in tissue sections. Promega Notes 72:7-9
Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pasla ST, van Oers MH (1994) Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84:1415-1420
Leers MP, Kolgen W, Bjorklund V, Bergman T, Tribbick G, Persson B, Bjorklund P, Ramaekers FC, Bjorklund B, Nap M, Jornvall H, Schutte B (1999) Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis. J Pathol 187:567-572[Medline]
Negoescu A, Lorimier P, LabatMoleur F, Drouet C, Robert C, Guillermet C, Brambilla C, Brambilla E (1996) In situ apoptotic cell labeling by the TUNEL method: improvement and evaluation of cell preparations. J Histochem Cytochem 44:959-968
O'Brien MA, Moravec RA, Riss TL (2001) Poly (ADP-ribose) polymerase cleavage monitored in situ in apoptotic cells. Biotechniques 30:886-891[Medline]
Sanders EJ (1997) Methods for detecting apoptotic cells in tissues. Histol Histopathol 12:1169-1177[Medline]
Srinivasan A, Roth KA, Sayers RO, Shindler KS, Wong AM, Fritz LC, Tomaselli KJ (1998) In situ immunodetection of activated caspase-3 in apoptotic neurons in the developing nervous system. Cell Death Differ 5:1004-1016[Medline]
Tanaka M, Momoi T, Marunouchi T (2000) In situ detection of activated caspase-3 in apoptotic granule neurons in the developing cerebellum in slice cultures and in vivo. Dev Brain Res 121:223-228[Medline]
Webb PR, Wang KQ, ScheelToellner D, Pongracz J, Salmon M, Lord JM (2000) Regulation of neutrophil apoptosis: a role for protein kinase C and phosphatidylinositol-3-kinase. Apoptosis 5:451-458[Medline]
Willingham MC (1999) Cytochemical methods for the detection of apoptosis. J Histochem Cytochem 47:1101-1109
Wyllie AH (1992) Apoptosis and the regulation of cell numbers in normal and neoplastic tissues: an overview. Cancer Metast Rev 11:95-103[Medline]