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Protocol: 1. Culture cells in medium containing 10% FBS for 1 day. 2. Aspirate media. Add fresh 10% FBS media with (treated) or without (control) 2 µM Camptothesin and culture for 8 hours. 3. Aspirate media from cultures; wash cells with 1X PBS; aspirate. 4. Fix cells using 1 ml 1X PBS containing 3% araformaldehyde for 20 minutes at 4°C. 5. Wash 3 times for 5 minutes each with TBS/T at room temperature. 6. Aspirate, then incubate with 1 ml Blocking Buffer for 45–60 minutes. 7. Aspirate, then incubate with primary antibody (at the appropriate dilution in TBS/T containing 5% BSA) for 24 hours at 4°C. 8. Wash three times for 5 minutes each time with 1 ml TBS/T. 9. Incubate with biotinylated secondary antibody (diluted appropriately in TBS/T/5% BSA; 1:500 for secondary antibody from Vector's VectaStain ABC kit) for 1 hour at room temperature. 10. Wash 3 times for 5 minutes each with 1 ml of TBS/T. 11. Wash once for 5 minutes with 1 ml of TBS. 12. Incubate for exactly 30 minutes in 0.6% H2O2 (200 µl 30% H2O2 in 10 ml TBS). 13. Wash 3 times for 5 minutes each with 1 ml of TBS/T. 14. Incubate 1 hour with 0.5–1 ml ABC reagent at room temperature (2 drops solution A into 5 ml PBS, mix, then add 2 drops solution B, mix). 15. Wash 3 times for 5 minutes each with 1 ml of TBS. 16. Add 1 ml DAB reagent. Monitor reaction progress under microscope. Reaction should proceed for 10 minutes. 17. Terminate reaction by adding 1 ml of water. 18. Aspirate and wash once with 1 ml of water. 19. View cells in 6-well plate or mount coverslips. |
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ABC-DAB Method 1. Wash tissue sections in Tris-buffered saline (TBS) containing 0.1% Triton X-100. 2. Treat sections with freshly made 1% H2O2 (0.1 ml of 30% H2O2 in 3 ml TBS) for 30 minutes. 3. Wash the sections with TBS/0.1% Triton X-100, three times for 30 minutes each at room temperature. 4. Block nonspecific binding sites with 3% BSA in TBS/0.1% Triton X-100 for 30 minutes to 1 hour. 5. Incubate the sections with primary antibody diluted in 3% BSA/TBS/0.1% Triton X-100 overnight at 4°C. 6. Wash the sections in TBS/0.1% Triton X-100, three times for 10 minutes each at room temperature. 7. Incubate the sections in biotinylated anti-rabbit secondary antibody (for polyclonal primaries) or biotinylated anti-mouse secondary antibody (for monoclonal primaries) diluted in 1% BSA/TBS/0.1% Triton X-100 for 1 hour at room temperature. 8. Prepare avidin-biotin-peroxidase complex (ABC) solution (Vectastain ABC Kits, Vector Laboratories Inc., Burlingame, CA) and leave the solution at room temperature for at least 15 minutes. 9. Wash the sections in TBS/0.1% Triton X-100, three times for 10 minutes each at room temperature. 10. Incubate the sections in the ABC solution for 1 hour at room temperature. 11. Wash the sections in TBS/0.1% Triton X-100, three times for 10 minutes each at room temperature. 12. Incubate the sections in DAB solution until staining is optimal as determined by light microscopic examination. 13. Wash the sections in TBS, three times for 5 minutes each. 14. Mount the sections on gelatin coated slides and dry them at room temperature. 15. Dehydrate the sections sequentially in 50%, 70%, 95% and 100% ethanol for 2 minutes each, 50%:50% ethanol/xylenes for 2 minutes and 100% xylenes for 5 minutes. 16. Mount the coverslides using Permount.
Double Fluorescent Labeling Protocol 1. Wash tissue sections in TBS containing 0.1% Triton X-100. 2. Block nonspecific binding sites with 3% 3. Incubate the sections with a first primary antibody (e.g., phospho-specific rabbit antibody) diluted in 3% BSA in TBS/0.1% Triton X-100 overnight at 4°C. 4. Wash the sections in TBS/0.1% Triton X-100, three times for 10 minutes each at room temperature. 5. Incubate the sections with a second primary antibody from a different species than the first primary antibody (e.g., mouse) diluted as above, for 2 hours at 4°C. 6. Wash the sections in TBS/0.1% Triton X-100, three times for 30 minutes each at room temperature. 7. Incubate the sections in a fluorescent secondary antibody mixture containing fluorescence-labeled secondary antibody against the first primary (i.e., fluorescence-labeled anti-rabbit) and fluorescence-labeled secondary antibody against the second primary antibody (i.e., fluorescence-labeled anti-mouse) each at a dilution of 1:200 in 1% BSA/TBS/0.1% Triton X-100 for 1 hour at room temperature. Incubation chambers should be covered with foil paper to avoid exposure to light. 8. Wash the sections in TBS, three times for 30 minutes each at room temperature. 9. Mount and coverslip the sections using Gelvatol. Add a small drop of Gelvatol to sections. Carefully place coverslip on the drop(s), avoiding air bubbles. 10. The mounting media will set overnight (at 4°C) or within 2–3 hours at room temperature.
Gelvatol Preparation: a. Add 2.4 g of Polyvinyl Alcohol (Mol. Wt. 30,000–70,000) to 6 ml of glycerol. Stir well to mix. Add 6 ml of dH2O and leave for at least 2 hours at room temperature. b. Add 12 ml of 0.2 M Tris (pH 8.5). Heat to 50°C for 10 minutes with occasional mixing. After polyvinyl alcohol is dissolved, clarify by centrifugation (5000 xg) for 15 minutes. Collect supernatant liquid. c. Add DABCO (1,4-diazabicyclo [2.2.2] octane; Sigma Cat. #D2522) to 2.5% as antifade medium. Aliquot in microtubes and store at –20°C. Stocks of Gelvatol are stable at room temperature for several weeks after thawing. |
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Protocol: 1. Deparaffinize/hydrate sections: a) Incubate sections in three washes of xylene for 5 minutes each. b) Incubate sections in two washes of 100% ethanol for 10 minutes each. c) Incubate sections in two washes of 95% ethanol for 10 minutes each. 2. Wash sections twice in ddH2O for 5 minutes each. 3. Wash sections in PBS for 5 minutes. 4. For antigen unmasking, heat sections in 10 mM sodium citrate buffer (pH 6.0) for 1 minute at full power followed by 9 minutes at medium power. (Keep slides fully immersed in buffer and maintain temperature at or just below boiling.) Cool slides for 20 minutes after antigen unmasking. 5. Wash sections in ddH2O three times for 5 minutes each. 6. Incubate sections in 1% hydrogen peroxide for 10 minutes. 7. Wash sections in ddH2O three times for 5 minutes each. 8. Wash section in PBS for 5 minutes. 9. Block each section with 100-400 ml blocking solution for 1 hour at room temperature. 10. Remove blocking solution and add 100-400 ml diluted primary antibody to each section. (Dilute antibody in blocking solution.) Incubate overnight at 4oC. 11. Remove antibody solution and wash sections in PBS three times for 5 minutes each. 12. Add 100-400 ml secondary antibody, diluted in blocking solution, to each section. Incubate 30 minutes at room temperature. 13. If using ABC biotin/avidin method, make ABC reagent according to the manufacturer’s instructions and incubate solution for 30 minutes at room temperature. 14. Remove secondary antibody solution and wash sections three times with PBS for 5 minutes each. 15. Add 100-400 ml ABC reagent to each section and incubate for 30 minutes at room temperature. 16. Remove ABC reagent and wash sections three times in PBS for 5 minutes each. 17. Add 100-400 ml DAB reagent to each section and monitor staining closely. 18. As soon as the section turns brown, immerse slides in ddH2O. 19. If desired, counterstain sections in hematotoxylin for 10 seconds. 20. Wash sections in ddH2O two times for 5 minutes each. 21. Dehydrate sections: a) Incubate sections in 95% ethanol two times for 10 seconds each. b) Repeat in 100% ethanol, incubating sections two times for 10 seconds each. c) Repeat in xylene, incubating sections two times for 10 seconds each. 22. Mount coverslips. |
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Preparing Cell Lysates1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time. 2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold PBS. 3. Remove PBS and add 0.5 ml 1X ice-cold Cell Lysis Buffer plus 1 mM PMSF to each plate (10 cm2) and incubate the plate on ice for 5 minutes. 4. Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice. 5. Sonicate 4 times for 5 seconds each on ice. 6. Microcentrifuge for 10 minutes at 4°C, and transfer the supernatant to a new tube. The supernatant is the cell lysate. If necessary, lysate can be stored at –80°C. Immunoprecipitation 1. Take 200 µl cell lysate and add primary antibody; incubate with gentle rocking overnight at 4°C. 2. Add Protein A Agarose Beads (20 µl of 50% bead slurry). Incubate with gentle rocking for 1–3 hours at 4°C. 3. Microcentrifuge for 30 seconds at 4°C. Wash pellet 5 times with 500 µl of 1X Cell Lysis Buffer. Keep on ice during washes. 4. Resuspend the pellet with 20 µl 3X SDS Sample Buffer. Vortex, then microcentrifuge for 30 seconds. 5. Heat the sample to 95–100°C for 2–5 minutes. 6. Load the sample (15–30 µl) on SDS-PAGE gel (12–15%). 7. Analyze sample by Western blotting (see Western Immunoblotting Protocol). |
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Protein Blotting: 1. Treat cells by adding fresh media containing regulator for desired time. 2. Aspirate media from cultures, wash cells with 1X PBS, aspirate. Scrape cells into PBS and spin down to pellet. 3. Lyse cells by adding Cell Extract Buffer (one volume of cell pellet, or 100 ml per well of 6-well plate or 500 ml per plate of 10 cm2 plate). Freeze and thaw 3 times. Centrifuge lysate at microcentrifuge using top speed. (~14000 rpm). Keep the supernatant and discard the pelleted cell debris. 4. Add SDS Loading Buffer and heat to 95-100oC for 5 minutes, cool on ice. 5. Microcentrifuge for 5 minutes. 6. Load 5-20 ml onto SDS-PAGE gel (10 cm x 10 cm). Note: We recommend loading prestained molecular weight markers to verify electrotransfer. 7. Electrotransfer to nitrocellulose membrane.
Membrane Blocking & Antibody Incubations: Note: Volumes are for 10 cm x 10 cm of membrane. For different sized membranes, adjust volumes accordingly 1. Incubate membrane in 25 ml of Blocking Buffer for 1 hour at room temperature. 2. Wash 3 times for 5 min each with 15 ml of TBS/T. 3. Incubate membrane and primary antibody (at the appropriate dilution) in 10 ml Primary Antibody Dilution Buffer with gentle agitation overnight at 4oC. 4. Wash 3 times for 5 minutes each with 15 ml of TBS/T. 5. Incubate membrane with HRP-conjugated secondary antibody in 10 ml of Blocking Buffer with gentle agitation for 1 hour at room temperature. 6. Wash membrane as in step 4. 7. Proceed with detection. Detection of Proteins: 1. Remove the wash buffer and place the blot in a plastic bag or clean tray containing chemiluminescent working solution (0.125 ml/cm2) and peroxide (ECL detection method). 2. Rotate the bag or tray to allow the solution to cover the surface of the membrane for 1-5 minutes. 3. Remove blot from bag or tray and place it between two pieces of write-on acetate transparency film. Smooth over covered blot to remove air bubbles and excess substrate. Expose to X-ray film. An initial exposure of 10-60 seconds is recommended for film.
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