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CUNY Graduate Center, New York
1 Hunter College of the City University of New York
Although specific proteins in living cells can now be labeled routinely with Green Fluorescent Protein, indirect immunofluorescence (IIF) methods for fixed material remain in widespread use (e.g., 1). While relatively easy to apply, the standard IIF procedure is lengthy and, for blood cells and other cell types in suspension, the required attachment of the material to a glass substrate can result in differential adhesion or losses. In addition, the non-mammalian erythrocytes and clotting cells (2–5) studied in our laboratory undergo a variety of naturally occurring or experimentally induced alterations to cell morphology. For these cell types, the fixation and permeabilization methods that have produced our best IIF cytoskeletal labeling to date have not preserved the morphology of the living cells very well.
This work had three initial objectives: (a) developing improved methods for morphological preservation and permeabilization of non-mammalian erythrocytes and clotting cells prior to IIF; (b) combining such methods with rapid fluorescence pre-labeling of a mouse primary antibody (use of ZenonTM; 6) to eliminate steps including substrate attachment; and (c) testing the combined approach on cells studied by others, or previously unstudied. For objectives (a) and (b) we employed dogfish erythrocytes and thrombocytes (Mustelus canis), blood ark erythrocytes (Anadara ovalis), and horseshoe crab amebocytes (Limulus polyphemus), all of which contain a marginal band (MB) of microtubules. For (c) we tested sea urchin sperm (Arbacia punctulata) and dividing zygotes (Lytechinus pictus) with known microtubule organization, plus spider crab hemocytes (Libinia emarginata) not studied previously.
Dogfish erythrocytes were first employed in an experimental survey of variables to develop both sequential and simultaneous methods of rapid fixation and permeabilization (objective a). Standard aldehyde or methanol fixation had produced cross-linked hemoglobin (Hb) that blocked antibody access in our earlier studies, and complete detergent lysis prior to fixation distorted cell morphology. Our experiments produced a major advance: brief formaldehyde prefixation (1%, < 7 min) and detergent extraction (0.4% Triton X-100, 10 min) yielded partial Hb retention and superior preservation of erythrocyte morphology, yet also allowed IIF labeling. Similar results were obtained with erythrocytes treated simultaneously with 4% formaldehyde and 0.6% Brij 58 (10 min). The slow extraction rate observed with Brij (compared with Triton) minimized morphological distortion when cells were not pre-fixed.
These methods were then tested on erythrocytes, other blood cells, and additional cell types in combination with ZenonTM labeling (objectives b and c). Thrombocytes were pre-fixed in 1% formaldehyde in 3% non-pyrogenic NaCl ("saline," 
7 min), then extracted with 0.4% Triton X-100 in PEM (100 mM PIPES, 5 mM EGTA, 1 mM MgCl2, pH 6.8, 10 min). Other cell types were permeabilized and fixed simultaneously in PEM containing 4% formaldehyde, 0.6% Brij 58, plus precautionary protease inhibitors (Sigma P8340 cocktail + 10 mM TAME; amebocytes: TAME only). All preparations were washed in phosphate buffered saline, (PBS, pH 7.2), then incubated in PBS containing mouse monoclonal anti-
and anti-ß tubulins (Sigma T9026, T-4026, 1:1 mass mixture) pre-bound with ZenonTM Alexa Fluor 488 Fab (Z-25002, 1:1 by mass; Molecular Probes). Representative results using a Zeiss standard epifluorescence microscope and Nikon 950 digital camera are shown in Figure 1.
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How rapidly were microtubules labeled? Perfusion chambers were made on slides scratched so as to enhance flow while retarding cell movement. Fixed dogfish leucocytes were loaded into the chamber and individual identified thrombocytes were viewed during successive perfusion with 0.4% Triton X-100 in PEM (10 min), and PBS (wash). Upon subsequent perfusion with ZenonTM-labeled anti-tubulin, the thrombocyte MB became visible almost immediately (< 30 s; Fig. 1c, c').
The rapidity of the procedure led us to ask whether all steps might be readily performed by perfusion, while individual pre-selected cells were being continually observed. This possibility was tested with dogfish thrombocytes, which can be activated to initiate clotting functions, adhesion, and shape transformation in saline containing 25U/ml bovine thrombin and 20 mM CaCl2. Living, unactivated thrombocytes were loaded into the chamber and activated. Then all steps of pre-fixation and extraction, postfixation (4% formaldehyde in PEM, 10 min), wash (PBS), and immunolabeling were performed by perfusion. Microtubule labeling was again rapid and comparable in intensity to that in Figure 1 (c, c'), confirming feasibility.
These new methods constitute a major practical advance. They now enable us to monitor morphological preservation and examine the cytoskeleton of preselected, activating blood cells at specific stages by perfusion. This process is much more rapid and convenient than was previously possible working with these cells. In addition, for cell populations naturally existing in suspension, all steps can be performed while maintaining them in suspension, bypassing glass adhesion (Fig. 1). Other important advantages for non-mammalian blood cells are (a) greatly improved preservation of erythrocyte morphology; (b) little non-specific labeling without blocking; (c) sufficient signal intensity even against the labeling antibody background; and (d) reduction of processing time from 4 h to 1 h. The same rapid methods are effective with other cell types (Fig. 1, f, f'; g, g') and with multiple labeling (e.g., DAPI; Fig. 1g' inset), indicating that they will be useful to other researchers. As a by-product, they may also be of considerable value in designing time-constrained laboratory exercises for courses.
We thank David Burgess and Michelle Ng for sea urchin samples, and HHMI Undergraduate Biosciences Education Program 2002679, PSC-CUNY64249, and NSF9726771 for support.
Literature Cited
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M. Conrad, J. DeNobile, I. Chaikhoutdinov, D. Escribano, K.-G. Lee, and W. D. Cohen Cytoskeletal Organization of Limulus Amebocytes Pre- and Post-Activation: Comparative Aspects Biol. Bull., August 1, 2004; 207(1): 56 - 66. [Abstract] [Full Text] [PDF]
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R. M. Pielak, V. A. Gaysinskaya, and W. D. Cohen Cytoskeletal Events Preceding Polar Body Formation in Activated Spisula Eggs Biol. Bull., October 1, 2003; 205(2): 192 - 193. [Full Text] [PDF]
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