BRIEF REPORT |
Optimal Processing Method to Obtain Four-color Confocal Fluorescent Images of the Cytoskeleton and Nucleus in Three-dimensional Chondrocyte Cultures
Institute of Biomedical Engineering (NT-K,DF,MDB) and Department of Chemical Engineering (AB,MDB), Ecole Polytechnique, Montreal, Quebec, Canada
Correspondence to: Michael D. Buschmann, Department of Chemical Engineering, Ecole Polytechnique, PO 6079, Station Centre-ville, Montreal, QC, Canada H3C 3A7. E-mail: michael.buschmann{at}polymtl.ca
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Summary |
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Key Words: cartilage chondrocyte cytoskeleton confocal microscopy actin tubulin vimentin
CARTILAGE REPAIR, regeneration, and tissue engineering are fields of intensifying efforts where improved structural characterization of in vitro constructs and their development and responses to mechanical and biological stimuli are required. The chondrocyte cytoskeleton is of key interest in many of these studies, particularly those involving biological responses to mechanical loads, given the role of the cytoskeleton in mechanotransduction (Durrant et al. 1999). The cytoskeleton comprises a three-dimensional network consisting principally of the three proteins actin, vimentin, and tubulin, where the monomers of these proteins are non-covalently associated and organized into microfilaments (MF), intermediate filaments (vimentin IF in chondrocytes), and microtubule (MT) filaments, respectively (Trickey et al. 2004
). The arrangement of these three filamentous systems is dynamic, extremely labile, sensitive to changes of environmental milieu, and partly masked by soluble and liposoluble proteineous structures with distinct biochemical characteristics (Arcangeletti et al. 1997
). A limiting factor in the investigation of the structure and function of the cytoskeleton of cells and tissues at present resides in the quality of processing, labeling, and imaging methods because the cytoskeleton is poorly preserved by most traditional histological fixation methods. We investigate the effects of different fixation and permeabilization methods on the preservation of cytoskeletal structure using bovine articular chondrocyte cultures in three-dimensional agarose hydrogels where maintenance of chondrocyte phenotype has been previously demonstrated (Szuts et al. 1998
).
Chondrocytes were enzymatically isolated from the humeral head cartilage of young bovine shoulders and encapsulated in 6-mm diameter x 2-mm-thick agarose disks as described previously (Tran-Khanh et al. 2005). On day 15 of culture, agarose disks were vertically cut into 600-µm-thick slices with a vibratome in standard Hank's balanced salt solution (sHBSS) [1.29 mM CaCl2·2H2O, 5.37 mM KCl, 0.44 mM KH2PO4, 0.49 mM MgCl2·6H2O, 0.41 mM MgSO4, 136.89 mM NaCl, 0.34 mM Na2HPO4·7H2O, 5.55 mM glucose, 4.2 mM NaHCO3, 15 mM HEPES (C8H18N2O4S), pH 7.4]. Good cell viability (>95%) was confirmed in these slices using Calcein AM and ethidium homodimer (Molecular Probes; Eugene, OR) as previously described (Dumont et al. 1999
). The cytoskeletal stabilization medium subsequently used for fixation and permeabilization was a modified Hank's balanced salt solution (mHBSS): 136.9 mM NaCl, 5.36 mM KCl, 2 mM MgCl2, 0.336 mM Na2HPO4, 0.44 mM KH2PO4, 4 mM NaHCO3, 2 mM EGTA, 5.55 mM D-glucose, 0.11% w/v 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5. This buffer was chosen for its low pH, chelation of calcium, and presence of magnesium, which aids in the preservation of cytoskeletal structures (Bacallao et al. 1995
).
We tested three families of processing methods on these chondrocyte-laden slices: (a) sequential fixation/permeabilization by 0.1% glutaraldehyde and/or 1% to 4% paraformaldehyde for 30 min at 37C, followed by permeabilization with 5% to 10% Triton X-100 for 20 min at 37C; (b) smooth fixation/permeabilization by 0.125% glutaraldehyde in the presence of 1% to 2% Triton X-100 for 20 min at 37C, prior to postfixation with 1% glutaraldehyde or 4% paraformaldehyde for 30 min at 37C; (c) simultaneous fixation/permeabilization with 0.3% to 0.6% glutaraldehyde and 2% to 5% Triton X-100 for 30 min at 37C or simultaneous fixation/permeabilization with 0.1% glutaraldehyde and 1% to 4% paraformaldehyde and 5% to 10% Triton X-100 for 30 min at 37C. An autofluorescence block was performed on the same day or on the following day by incubating slices in 5 mg/ml NaBH4. Antibody penetration was then facilitated by digesting slices in 200 mU/ml chondroitinase ABC and 400 mU/ml keratanase as described previously (Langelier et al. 2000). Nonspecific antibody-binding sites were blocked by incubation in 10% goat serum (Sigma; St Louis, MO) and 0.01% Tween-20 in mHBSS for 2 hr. The buffer in the following steps was mHBSS containing 1% goat serum and 0.05% Tween-20 with agitation. Blocked slices were incubated with 10 µg/ml monoclonal anti-tubulin-ß (Chemicon; Temecula, CA) for 16 hr at 4C, followed by three 20-min washes. Slices were then incubated in 10 µg/ml polyclonal goat anti-mouse coupled to Alexa Fluor 405 (Molecular Probes) for 4 hr and washed four times during 20 min. The buffer in the following steps was mHBSS containing 1% BSA and 0.05% Tween-20 with agitation. Tubulin-labeled slices were labeled for actin and vimentin with 0.2175 µg/ml Alexa Fluor 488phalloidin (Molecular Probes) and 1.43 µg/ml monoclonal antibody anti-vim-Cy3 (Sigma) and washed three times during 20 min. Nuclear DNA was stained by first removing nonspecific RNA signal by 2-hr incubation with 1 mg/ml RnaseA (Qiagen; Mississauga, Canada) in PBS and 1% BSA followed by DNA staining with 3 µM of TOTO3 (Molecular Probes) in PBS for 2 hr. These four-color-stained slices were immersed for 30 min in glucose oxidase/catalase (GOC) anti-fading reagent and mounted with Mowiol on slides with coverslips N° 1.5 as described previously (Langelier et al. 2000
). Fluorescence imaging of chondrocytes in 600-µm agarose slices was performed using a LSM 510 META Axioplan 2 confocal laser scanning microscope with C-Aprochromat x40/1.2 water-immersion objective (Carl Zeiss; Jena, Germany). Laser lines used were 488-nm argon laser, 543- and 633-nm heliumneon lasers, and 810 nm- (two-photon) pulsed titanium sapphire laser (VerdiV10/Mira 900; Coherent, Santa Clara, CA) using dichroic HFT UV/488/543/633 for conventional laser lines and HFT KP650 for the pulsed laser. The meta function was used to select filter and dichroic mirror configurations that minimized overlap from the four different fluorochromes. Images were recorded at 810-nm excitation using a BP 390465 IR band pass filter for Alexa Fluor 405, at 488-nm excitation using a BP 510520 IR band pass filter for Alexa Fluor 488, at 543-nm excitation using a BP 565615 IR pass filter for Cy3, and at 633-nm excitation and a BP 644676 meta filter for TOTO-3. The pinhole size was adjusted to obtain the optimal spatial resolution and high-magnification images were recorded with a 0.45-µm z-step and 0.08 µm x/y pixel size. Calcein-loaded chondrocytes under the viability test were also imaged as described above for Alexa Fluor 488 but using a water- immersion objective IR-Achroplan x63/0.9 dipped directly in sHBSS. Image stack images were deconvolved with Huygens2 software and presented with Imaris 4.0 software (Bitplane AG; Zurich, Switzerland). Images are representative of at least three sets of individual chondrocytes.
Because multiple stains have a tendency to overlap spectrally and spatially, especially when four stains are used, and cellular components of interest as microtubule and vimentin network are known to colocalize, we chose the Alexa Fluor 405, Alexa Fluor 488, Cy3, and TOTO-3 dyes to minimize overlap. A rigorous lambda mode analysis using the META function provided excitation and emission spectra of each dye to optimize filter configuration (Dickinson et al. 2001) and ensure a distinct separation of signals in the quadruple-stained specimens (Figure 1). Confocal z-series through representative chondrocytes grown in agarose showed a well-organized distribution of MT, MF, and vimentin IF (Figure 2) comparable to monostains previously described in situ and in agarose (Idowu et al. 2000
). Because several previous studies have shown that precipitating fixatives such as acetone or alcohols produce cellular shrinkage and disruption with poorly preserved cytoskeletal organization (Bacallao et al. 1995
), these precipitating fixatives were not examined in our study.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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