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| MICROTUBULE STAINING | ||
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Microtubule Staining
1. Add ~60µls of 1mg/ml Polylysine to each round cover slip (12 mm dia) 2. Let stand at room temp. for 20-30 min. 3. Remove excess fluid with aspirator shouldn't have to rinse 4. Meanwhile : assemble Microtubules ~30 min. at 37 C stabilize with taxol ~15 min. at 37 C 5. Make up 100 µls of 45 mg/ml EGS in DMSO 6. Preheat 8 sample tubes containing 900 µls of 1x BRB80 at 37 C for 10 min. 7. Add 100 µls of EGS stock to one tube 8. Add 4 µls of assembled/stabilized MT's to tube containing EGS using cut off pipette tip 9. Incubate for 15 min. at 37 C 10. Meanwhile : locate 6 specially constucted 15ml Corex tubes containing plugged bottoms add ~7µls of BRB80 to the top of the plugs that fit in these tubes place one round cover slip (polylysine side up) on each plug add 300µls of BRB80 to each corex tube drop plugs into tubes , making sure that each plug is surrounded by but not covered w/ BRB80 11. Perform a series of 10 fold dilutions allowing for (2) complete 1:10 dilutions (2) complete 1:100 dilutions and... (2) complete 1:1000 dilutions * Note : Use cut off pipette tips 12. Add all 1000 µls of each dilution to its respective corex tube (just above cover slip) 13. Spin Samples 10 K for 45 min. at 23 C JS 13.1 swinging bucket rotor Beckman After spin ... 14. remove cover slips from tubes and immediately place in Methanol kept at -20 C for 5'. *Note : care must be taken to keep cover slips in correct orientation and in order. 15. Perform 3 washes in 1xPBS + Triton for 30 sec. each then... lay out onto a large petri-dish with numbered positions ( MT side up ) 16. Add 20 µls of the primary antibody 17. Let stand at room temp. for 20 min. 18. Remove excess fluid with aspirator 19. Perform 3 washes in 1xPBS + Triton for 30 sec. each 20. Add 20 µls of the secondary antibody 21. Let stand at room temp. for 20 min. 22. Remove excess fluid with aspirator 23. Perform 3 washes in 1xPBS + Triton for 30 sec. each 24. Place cover slips "face-down" onto microscope slide containing 2.5 µls of mounting media. *Note : use care to minimalize cover slip movement when laying on mounting media 25. Seal with fingernail polish. 2 Possible Antibody series : 1 2 primary antibody primary antibody YOL 134 1:200 dilution in sol'n F Anti-alpha tubulin 1:500 dil. in sol'n F secondary antibody secondary antibody Goat anti Rat FL 1:300 dilution in sol'n F Goat anti mouse RH. 1:200 dil. in sol'n F * Note : Final conc. of 0.5% Glutaraldehyde (5 min. at 23 C)can be substituted for EGS fixation. 62.5 µls of 8% Glutaraldehyde stock + 937.5 µls of 1x BRB80 1x PBS Buffer 1 liter = 8 g NaCL 0.2 g KCL2 1.14g Na2HPO4 0.2 g KH2PO4 pH 7.3 add Triton stock = 0.1% Final conc. add Na Azide = 0.02% Final conc. |
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