Tung Ling L Chen Peiyin Wang Seung S Gwon Nina W Flay and Barbara M Vertel

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1. Introduction

Proteoglycans undergo numerous synthetic and processing events as they progress through the exocytic pathway. In cells that express genetically engineered constructs encoding proteoglycans, immunolocalization is a useful approach in identifying specific intracellular compartments involved in their processing and trafficking.

2. Materials

2.1. Expression of Target Proteoglycan (Cell Culture and Transfection)

1. Proteoglycan constructs packaged into vectors suitable for expression in target cells (1,2). In our experiments, pcDNA3 and its derivatives (Invitrogen) were used. FLAG and 6xHis epitope tags were engineered at the C-terminus.

2. Mammalian cell lines: Chinese hamster ovary (CHO) cells, COS-1 cells, and HeLa cells were used.

3. Culture medium: Ham's F12 medium (for CHO, Life Technologies) or Dulbecco's Modified Eagle's Medim High Glucose Pyruvate (HG-DMEM, for COS-1 and HeLa, Irvine Scientific) with 10% fetal bovine serum (Atlanta Biologicals) and 1% antibiotic-antimycotic solution (Life Technologies).

4. Trypsin-EDTA solution (Sigma, cell culture grade).

5. Tissue culture dishes.

6. Sterilized cover slips: No. 1 cover slips were prepared by soaking in 95% ethanol for at least 10 min, followed by rinsing with sterile distilled water.

7. SuperFect transfection reagent (Qiagen).

8. Opti-MEM (Life Technologies).

9. Phosphate buffered saline (PBS).

2.2. Immunofluorescence Localization of Expressed Proteoglycan

1. Fixatives: 100% methanol, stored at -20°C or 2-4 % paraformaldehyde in PBS.

3. Normal goat serum (NGS).

4. Primary antibody: M2 anti-FLAG antibody (Eastman Kodak), Penta-His or Tetra-His antibody (Qiagen).

5. Secondary antibody: FITC-conjugated goat IgG anti-mouse IgG (Jackson Labs).

7. Mounting medium (3): 15% Vinol 205 polyvinyl alcohol (w/v, see Note 1), 33% glycerol (v/v), 0.1% azide, pH 8.5. Dissolve 20 g of Vinol 205 polyvinyl alcohol in 80 mL of 0.1 M Tris, 0.1 M NaCl, pH 8.5, for 16 h with stirring. Add 40 mL of glycerol and 1.2 mL of 10% Na azide with continued stirring for another 16 h. Pellet undissolved particles at 20,000g for 20 min. Aliquot the viscous supernatant and store at -20°C. Use a defrosted aliquot stored at 4°C as your working solution.

2.3. Ultrastructural Localization of Expressed Proteoglycan

1. Paraformaldehyde-lysine-periodate fixative (4):

a. Solution A: 0.1 M lysine-0.05 M phosphate buffer, final pH = 7.4. Dissolve 1.827 g of lysine HCl in 50 mL of distilled water. Adjust to pH 7.4 with 0.1 M Na2HPO4. Bring the final volume to 100 mL with 0.1 M phosphate buffer, pH 7.4. Osmolarity should be approx 300 mo. Store at 4°C.

b. Solution B: 20% paraformaldehyde. Mix 10 g of paraformaldehyde in 50 mL of distilled water, heating in a 60°C water bath with stirring. Slowly add 1-3 drops of 1 N NaOH until the solution clears. Store at -20°C. Spin or filter to remove debris before use.

To make 10 mL of fixative, mix 7.5 mL of solution A with 1 mL of solution B and 1.5 mL of distilled water. Add 21.4 mg of NaIO4. Final composition is 0.01 M NaIO4, 0.075 M lysine, 0.0375 M phosphate buffer, 2% paraformaldehyde. Upon mixing solutions A and B, the pH will decrease from 7.4 to approx 6.2. The fixative is used at this lower pH and needs to be made fresh directly before use.

2. Wash and permeabilization solution: 0.05% saponin in PBS.

3. Blocking solution: 10% NGS/0.06% glycine/0.05% saponin/PBS.

4. Primary antibody: M2 anti-FLAG, diluted 1/100 in PBS with 5% NGS and 0.05% saponin.

5. Rabbit IgG anti-mouse IgG, diluted 1/25 in PBS with 5% NGS and 0.05% saponin.

6. Peroxidase-conjugated goat IgG anti-rabbit IgG Fab fragments (Jackson Labs), diluted 1/50 in PBS with 5% NGS and 0.05% saponin.

7. Diaminobenzidine (DAB): Tare a 15-mL polystyrene tube, weigh out DAB, and dissolve in DAB buffer to generate a 0.2% solution—e.g., weigh out 10 mg of DAB and dissolve in 5 mL of DAB buffer. (see Note 2.)

9. Hydrogen peroxide (30% solution, Sigma).

10. Sucrose-containing cacodylate buffer: Mix 0.2 MNa cacodylate, pH 7.4, and 60% sucrose to make 0.1 M Na cacodylate containing 6% sucrose.

11. Glutaraldehyde (grade I, 25% aqueous solution, Sigma). (see Note 2.)

12. Osmium tetroxide (2% aqueous solution, Electron Microscopy Sciences) (see Note 2).

13. Potassium ferrocyanide [K4Fe(CN)6 . 3H2O, Sigma]. (see Note 2.)

14. Ethanol.

15. Hydroxypropyl methacrylate (HPMA, Electron Microscopy Sciences).

16. Epon with catalyst:

a. tEpon 812 (Tousimis Research Corp.).

b. Nadic methyl anhydride (NMA, Tousimis Research Corp.).

c. Dodecenylsuccinic anhydride (DDSA, Tousimis Research Corp.).

d. Tri-dimethylamino methyl phenol (DMP-30, Tousimis Research Corp.).

To make 50 mL of mixture, pour 21 mL of NMA into a 100 mL plastic beaker with a volume marker, add 3 mL of DDSA using a syringe, then add tEpon 812 to the 50-mL mark. Stir with a wood stick for 15 min, taking caution to avoid bubbles. Add 0.75 mL of DMP-30 with a syringe, and stir well for another 10 min. Unused portions may be stored in syringes at -20°C for later use. (see Note 2.)

17. Lead citrate: The lead citrate solution is prepared and used according to Reynolds (5). All glassware must be washed with 3% HCl and thoroughly rinsed with double-distilled (dd) water in advance. Fill a 50-mL volumetric flask with 30 mL of dd water. Add 1.33 g of Pb(NO3)2 and 1.76 g of trisodium citrate. Shake continuously for 1 min. The contents will appear milky. Let stand for an additional 30 min with intermittent shaking. This is necessary for the complete conversion of reagents into lead citrate. Adjust to pH 12 by the slow addition (with inversion of the flask) of 1.0 N NaOH. The proper pH is reached just at the point when the solution clears (after the addition of about 8 mL of 1.0 N NaOH). Adjust to the final volume of 50 mL using dd water. Filter with Whatman's #2 filter paper. Store the solution at room temperature in a tightly capped amber glass bottle. The solution should not be jarred; allowing it to stand undisturbed enables small particles to settle to the bottom and improves the quality of the stain.

3. Methods

3.1. Expression of Target Proteoglycan (Cell Culture and Transfection)

1. Seed 1.1 x 106 CHO cells onto a 100-mm culture dish containing sterilized cover slips 36-40 h before transfection (see Notes 3 and 4). For ultrastructural studies, seed 1 x 105 cells onto a 35-mm culture dish.

2. Incubate in a 37°C incubator with 5% CO2. For CHO cells, the dishes should be 80% confluent on the day of transfection.

3. In a polystyrene tube, dilute 5 ^g of DNA into 150 ^L of Opti-MEM (contains no serum or antibiotics). Mix solution.

4. Add 10 ^L of SuperFect transfection reagent to the DNA solution (see Note 5). Mix solution.

5. Incubate the samples for 5-10 min at room temperature (20-25°C) to allow complex formation.

6. While complex formation takes place, set up 35-mm dishes with 1 mL of Opti-MEM and transfer one cover slip with attached cells to each dish.

7. Add 1 mL of complete medium containing serum and antibiotics to the reaction tube containing the transfection complexes. Mix by pipetting up and down twice, and immediately transfer the total volume to the cover slips in the 35-mm dishes.

8. Incubate the cells with the complexes for 2 h at 37°C in an atmosphere of 5% CO2.

9. Remove medium containing the remaining complexes from the cells. Wash cells once with PBS.

10. Add new complete medium. Incubate cells for additional hours as each experiment requires (usually 24-48 h).

3.2. Immunofluorescence Localization of Expressed Proteoglycan

1. Prior to fixation, wash cells with PBS 2 times.

2. Fix with cold methanol for at least 20 min, or with room-temperature paraformaldehyde for 15 min.

mlnisggrecau

Fig. 1. Immunofluorescence localization of expressed "miniaggrecan" (containing the chicken aggrecan N-terminus with the signal sequence and G1 domain, a segment of the chon-droitin sulfate attachment region, the G3 domain, and a 6xhis epitope tag) and the Golgi enzyme, ST. Transfected CHO cells exhibit miniaggrecan localization within the perinuclear Golgi complex (B). The region is further identified as the Golgi complex by the co-transfection and localization of the ST Golgi enzyme (C). The corresponding phase micrograph shown in (A) contains an expressing cell that exhibits positive immunofluorescence localized to the Golgi subcellular compartment, and nonexpressing cells that serve as useful negative controls. Miniaggrecan was detected using the mouse monoclonal Tetra-His Antibody and FITC-goat IgG anti-mouse IgG. ST was localized using polyclonal anti-ST antibodies and Texas Red-goat IgG anti-rabbit IgG. The calibration bars of Figs. 1 and 2 represent 10 ^m.

3. Permeabilize the paraformaldehyde-fixed cells with 0.1% NP-40 for 15 min. (Skip this step if the cells were fixed with methanol.)

4. Rinse with PBS.

5. Block the fixed samples with 15% NGS at 37°C for 15 min.

6. After removing NGS, add primary antibody to the sample (anti-FLAG was used at 0.04 mg/mL, Tetra-and Penta-His were used at 0.01 mg/mL) and incubate for 2 h at 37°C.

7. Wash with PBS for 1 h with 5-6 changes.

8. Incubate with FITC-conjugated goat IgG anti-mouse IgG (0.2 mg/mL) for 1 h at 37°C.

9. Wash with PBS for 1 h with 5-6 changes.

10. If double-fluorescence localization is desired, repeat steps 4-7 using selected rabbit polyclonal antibodies and Texas Red-conjugated goat IgG anti-rabbit IgG (see Note 6).

11. Mount samples on microscope slides with mounting medium.

12. Observe samples with a microscope equipped with phase-contrast (or differential interference-contrast) and incident-light fluorescence optics (see Notes 7 and 8). Document with conventional photographic camera or capture images using digital camera (see Figs. 1 and 2).

3.3. Ultrastructural Localization of Expressed Proteoglycan

1. Wash cells with PBS 2 times.

2. Fix with paraformaldehyde-lysine-periodate fixative for 45 min at room temperature (see Notes 9 and 10).

3. Wash with PBS 2 times.

4. Incubate 15 min with 0.05% saponin/PBS to permeabilize cells.

5. After most of the wash solution is removed, use Kimwipes to dry the edge of culture dish. This step generates surface tension around the central area of the dish so that as little as 50 ^L of blocking reagents (and later, antibody solutions) is required for covering the area during incubation. Incubate in blocking reagent for 20 min.

Fig. 2. Colocalization of G1-aggrecan (containing the chicken aggrecan N-terminus with the signal sequence and G1 domain, a segment of the chondroitin sulfate attachment region, and a 6xhis epitope tag) and concanavalin A. CHO cells transfected with G1-aggrecan exhibit protein localized principally in regions throughout the cytoplasm (B). Identification of these cytoplasmic regions as the ER is established by the colocalization of FITC-concanavalin A, a lectin that recognizes mannose-rich oligosaccharides in the ER (C). Two expressing cells and segments of several nonexpressing cells are revealed in the corresponding phase micrograph (A) and by the concanavalin A localization (C). The nonexpressing cells serve as negative controls for G1-aggrecan localization, which was accomplished using the mouse monoclonal Penta-His Antibody and Texas Red-goat IgG anti-mouse IgG. Reproduced with permission from Wang, P. W., Chen, T.-L., Luo, W., Zheng, J., Qian, R., Tanzer, M. L., Colley, K., and Vertel, B. M. (1999) Immunolocalization of 6xHis-tagged proteins in CHO cells with QIA express Anit-His Antibodies. Qiagen News 1, 3-6. (6). Used with permission.

6. Wash with 0.05% saponin/PBS 2 times before adding primary antibody.

7. Run Kimwipes around edge of culture dish to prepare the surface for antibody addition and incubate in primary antibody for 1.5 h at 37°C.

8. Wash 6-7 times over 45 min with 0.05% saponin/PBS.

9. Run Kimwipes around edge of culture dish to prepare the surface for antibody addition and incubate in rabbit IgG anti-mouse IgG for 1.5 h at 37°C.

10. Wash 6-7 times over 45 min with 0.05% saponin/PBS.

11. Run Kimwipes around edge of culture dish to prepare the surface for antibody addition and incubate in peroxidase-conjugated goat anti-rabbit FAB for 1.5 h at 37°C.

12. Wash 6-7 times over 45 min with 0.05% saponin/PBS.

13. Wash 3 times over 5 min in DAB buffer.

14. During wash, prepare DAB solution.

15. Add 1.3 mL of DAB solution per dish and incubate 10 min on a shaker.

16. Add 1.5 ^L of hydrogen peroxide for each dish, cover the dish, and incubate for an additional 5-10 min. Monitor color change using an inverted microscope and when reaction is sufficient, stop by removing DAB solution (see Note 11).

17. Wash 3 times over 10 min with DAB buffer.

18. Wash for 10 min with 1/1 mixture of DAB buffer and sucrose-containing cacodylate buffer.

19. Wash with sucrose-containing cacodylate buffer 2 times, 10 min each.

20. Fix with 2% glutaraldehyde in 0.1 M cacodylate buffer containing 6% sucrose for 30 min at room temperature.

21. Wash with sucrose-containing cacodylate buffer 2 times, 10 min each.

22. Fix with 1% OsO4/1.5% KFe4(CN)6 in 0.1 M cacodylate buffer containing 6% sucrose for 45 min in the dark and cold.

23. Wash with sucrose-containing cacodylate buffer 4 times, 15 min each.

24. Fix with 1% tannic acid in 0.1 M cacodylate buffer containing 6% sucrose for 15 min.

25. Wash with sucrose-containing cacodylate buffer 2 times, 10 min each.

26. Wash with water 3 times over 10 min.

27. Dehydrate through 50% ethanol, 10 min; 75% ethanol, 10 min; 90% ethanol, 10 min; 90% HPMA, 3 times over 15 min; 95% HPMA, 15 min; 97% HPMA, 15 min (see Note 12).

28. Exchange through a series of HPMA/tEpon solutions: 2/1 HPMA/tEpon, 15 min; 1/1 HPMA/tEpon, 30 min; 1/2 HPMA/tEpon, 30 min. Add HPMA/tEpon mixture to dish, cover, and mix continuously.

29. Exchange into Epon with catalyst over 30 min with 3 changes.

30. Drain off excess Epon with catalyst to leave just enough to cover the cell layer.

31. Infiltrate overnight at 37°C in oven with dessicant. Be sure dishes in the oven are flat, and cover them with a single layer of foil into which small holes have been poked to allow residual dehydrating agents to evaporate off.

32. Transfer dishes to 60°C oven for polymerization, keeping the dishes flat to maintain a uniform depth of Epon polymer on the cells. Two days are required for complete polymerization.

33. Upon completion of polymerization, break the culture dish to recover the embedded cells in a thin layer of polymerized Epon (see Note 13). Cut out a small piece (<1 mm each side) to be mounted (using epoxy or Krazy Glue) on a block suitable for sectioning. A light microscope can be used to help in selecting optimal areas for mounting.

34. Ultrathin sections are collected onto electron microscope grids. Cells may be counter-stained briefly with lead citrate before viewing under the electron microscope. For lead citrate counterstaining, prepare a Petri dish chamber with parafilm on the bottom. Add moist NaOH pellets to the chamber in order to sequester CO2 and prevent the formation of lead carbonate precipitate. For counterstaining, drop the lead citrate solution from a syringe with a filter onto the parafilm. Float each grid, sample side down, onto separate drops. Cover the chamber with aluminum foil to protect against light and CO2 contamination. Remove excess stain from the grids by dipping them through a series of double-distilled water rinses and allow the grids to dry before viewing under the electron microscope.

4. Notes

1. Vinol 205 polyvinyl alcohol (also called Airvol 205) is available as a sample on request from Air Products and Chemicals, Inc. (Allentown, PA). Defrosted working solutions are stable for 6 mo at 4°C.

2. Toxic compounds used in this procedures include DAB, cacodylate, osmium tetroxide, glutaraldehyde, and the dehydration solutions and nonpolymerized embedding materials. Handle the solutions in the hood with gloved hands. Dispose as chemical waste. DAB can be detoxified by treatment with chlorox.

3. The initial cell density required to seed the cover slips varies among cell types used for trans-fection. The major factors to be considered are cell growth rate, the time required for cells to attach to the cover slips, and the survival rate after transfection. For example, CHO cells can grow directly on the glass surface of cover slips, but require 36-40 h to attach and spread well, while COS and HeLa cells attach and spread better on gelatinized carbon-coated cover slips, and do so by 24 h. The COS and HeLa cells are less sensitive to SuperFect transfection reagent, and so 70% confluence is the optimal density at the time of transfection. Cell density adjustments may need to be made to allow for differences in the toxicity of SuperFect lots.

4. Gelatinized carbon-coated coverslips can be used for cells that do not attach well to uncoated glass surfaces. A carbon coat is applied to glass cover slips using carbon rods

(Ted Pella) in a vacuum evaporator (Denton, DV502) run under standard conditions. Carbon-coated cover slips are stored in 95% ethanol and treated with 1% gelatin before use.

5. The optimal volume (^L) of SuperFect reagent and the ratio of SuperFect volume to the quantity of DNA (^g) may vary depending on the specific cell type and DNA construct.

6. Compartment-specific antibodies and fluorophore-conjugated lectins can be used to help identify intracellular compartments involved in trafficking of proteoglycans. For example, concanavalin A, a lectin that reacts with mannose-rich oligosaccharides added co-translationally to glycoproteins in the endoplasmic reticulum (ER), can be used as a marker for the ER. Co-transfection with a construct that encodes the Golgi enzyme sialyltransferase (ST) has been used in our experiments to identify the Golgi complex (6). In this case, expressed ST was localized with polyclonal antibodies against ST. Alternatively, antibodies specific for Golgi complex proteins, such as TGN38, may be used to identify the trans-Golgi network.

7. When observing immunostained cells under the fluorescence microscope, it is important to distinguish real signals from nonspecific background. An overall high level of cellular fluorescence usually suggests a background problem. In transfection experiments, the nontransfected (therefore, nonexpressing) cells serve as a convenient internal negative control for immunostaining (see Figs. 1 and 2).

8. The background problem in immunostaining reactions can usually be reduced by diluting the primary and/or secondary antibody concentrations and by modifying incubation times.

9. If the final concentration of paraformaldehyde used in the fixative is increased to improve ultrastructure, the effect on antigenicity must also be determined.

10. The protocol for preembedment immunoperoxidase localization is a modification (7) of the method described by Brown and Farquhar (8).

11. DAB color reaction should be stopped when the reaction becomes saturated and starts to extend into peripheral structures.

12. The protocol for embedding monolayer cell cultures is according to Brinkley et al. (9).

13. For ultrastructural localization studies, the monolayer of cells is best cultured in dishes with thin walls because these plastic dishes can be pried off easily to release the polymerized Epon-containing embedded cells.

Acknowledgements

Research support was provided by National Institutes of Health grants DK28433, AR45909, and grants from the Arthritis Foundation. We are grateful to Drs. Marvin Tanzer and Wei Luo (Univ. Connecticut Health Center) for providing aggrecan constructs and Dr. Karen Colley (Univ. Illinois College of Medicine, Chicago) for providing antibodies and constructs for sialyltransferase.

References

1. Zheng, J., Luo, W. and Tanzer, M. L. (1998). Aggrecan synthesis and secretion. A paradigm for molecular and cellular coordination of multiglobular protein folding and intracellular trafficking. J. Biol. Chem. 273, 12,999-13,006.

2. Luo, W., Kuwada, T. S., Chandrasekaran, L., Zheng, J., and Tanzer, M. L. (1996). Divergent secretory behavior of the opposite ends of aggrecan. J. Biol. Chem. 271, 16447-16450.

3. Dahdal, R. Y. and Colley, K. L. (1993) Specific sequences in the signal anchor of the galactoside-2,6-sialyltransferase are not essential for Golgi localization. J. Biol. Chem. 268, 26,310-26,319.

4. McLean, I. W. and Nakane, P. K. (1974) Periodate-lysine-paraformaldehyde fixative: a new fixative for immunoelectron microscopy. J. Histochem. Cytochem. 22, 1077-1083.

5. Reynolds, E. S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212.

6. Wang, P. W., Chen, T.-L., Luo, W., Zheng, J., Qian, R., Tanzer, M. L., Colley, K., and Vertel, B. M. (1999) Immunolocalization of 6xHis-tagged proteins in CHO cells with QI A express Anti-His Antibodies. Qiagen News 1, 3-6.

7. Vertel, B. M., Velasco, A., LaFrance, S., Walters, L., and Kaczman-Daniel, K. (1989) Precursors of chondroitin sulfate proteoglycan are segregated within a subcompartment of the chondrocyte endoplasmic reticulum. J. Cell Biol. 109, 1827-1836.

8. Brown, W. J. and Farquhar, M. G. (1984) The mannose-6-phosphate receptor for lysosomal enzymes is concentrated in cis Golgi cisterna. Cell 36, 295-307.

9. Brinkley, B. R., Murphy, P., and Richardson, C. L. (1967) Procedure for embedding in situ selected cells cultured in vitro. J. Cell Biol. 35, 279-283.

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