Practica # 12 INMUNOTINCION CON FLUORESCENCIA INTRODUCCIN La tecnologa de la inmunotincion esta siendo muy empleada en la investigacin cientfica. Gracias al uso de un anticuerpo especfico se puede detectar una protena (antigeno), dentro de una clula, ubicar su localizacin celular o interaccin con otra protena, este ultimo cuando se usa dos anticuerpos diferentes. En la inmunodeteccin se usa generalmente dos tipos de anticuerpos: el primario, que es especifico para un tipo de antigeno (por ejemplo anti-p53 hecho en ratn) y un segundo anticuerpo que reconoce al primero (anti-Ig de ratn) y adems esta conjugado con molculas fluorescentes como fluorescena (verde) o Rhodamina (rojo). Esta tecnologa consiste primero en fijar la clula sobre un portaobjeto, permeabilizar e incubar con un primer y finalmente con un segundo anticuerpo.

En la figura 1 podemos observar El ncleo celular y el Citoesqueleto formado por microtubulos. Clulas macrfagos fueron fijados sobre una cubreobjeto recubierto (coated) con una delgada pelcula de polilisina. Despus de una permeabilizacion con triton X100, se uso anti-tubulina como primer anticuerpo y anti anti mouse conjugado con fluorescena, como segundo anticuerpo. El ncleo fue teido con Hoesch. Vista representativa con microscopio de fluorescencia vista con objetivo 60X (fuente: produccin propia, elaborada en los laboratorios de Harvard Medical School)

OBJETIVO: - Desarrollar la tecnologa de la Inmunotincion PROCEDIMIENTO. Se describe el protocolo utilizado en Dpto de Biologa Molecular de Harvard medical School. Cell Fixation 1. Incubate 4% Paraformaldehyde in PBS for ten minutes 2. Then take six well plate from incubation and remove all medium from the well 3. Then administer 500 microliters paraformahyde solution to the coverslips in the wells 4. Allow wells to incubate for thirty minutes 5. Take coverslips from the wells and put into a humid chamber* 6. Then remove excess paraformaldehyde from the coverslips 7. Add 500 microliters of NH4CL 50 mM in PBS to the coverslips to neutralize reaction allow to sit for five minutes 8. Remove the NH4CL 9. To wash coverslips add 1 mL PBS four times separately over a entire duration of 5 minutes 10. Add 200 microliters blocking solution* allow to sit for thirty minutes 11. Remove blocking solution 12. Add 200 microliters of primary antibody* and allow to sit for 1-2 hours minimum 13. Remove primary antibody 14. Wash coverslips add 1 mL PBS four times separately over a entire duration of 5 minutes 15. Add 200 microliters secondary antibody* and allow it sit for 45mintutes to an hour 16. Remove all second antibody 17. Wash with PBS three times for five minutes each (fifteen minutes). 18. Use pipet tips to lift coverslips hold using hand making sure not to touch cells 19. Clean reverse side of coverslip with porous paper 20. Pipet off any remaining PBS 21. Each individual slide need two separate drops of GVA 22. Place coverslip on drop and press ensuring not to let slide slip 23. Place in dark area until ready to finish 24. All nail polish to edges of coverslip for firm hold *Humid Chamber 1. Discard parafilm cover from sporous paper 2. Soak the paper with water

3. Replace paraflim cover with new flim *Blocking Solution 1. Add 630 microliters PBS to 2. 70 microliters BSA 10% in PBS and 3. 3.5 microliters Triton X100 20% *Primary Antibody 1. Add 200 microliters blocking solution to individual tubes 2. Use ratio for primary antibody and add appropriate amount of antibody to respective tubes 3. RassF1A 1:200 Tubulin Alpha/Beta 1:100 *Secondary Antibody 1. Add 200 microliters blocking solution to individual tubes 2. Use ratio for secondary antibody and add appropriate amount of antibody to respective tubes 3. Add 1:1000 Dapi to solution 4. Actin = Phalloidin 1:80 RassF1A = Mouse Green 1:200 Tubulin Alpha/Beta = Rabbit Red 1:200 Anti-Flag = 1:1000 Alternate Methodology 1: 1. Set up a coverslip clulture of an appropriate amount cell line 24 hrs prior to lab. This is best accomplished by dry sterilization of #1 coverslips which are subsequently placed in plastic tissue cultures plates. Cells are placed on the coverslips with sufficient media to cover and allowed to grow fo r 24 hrs. There should be sufficient cells to view comfortable, but they should not be crowded on the side 2. Remove coverslips from the culture plate and dip several times in a beaker of phosphate buffered saline (PBS) to rinse off the culture media. Drain, but do not allow to dry 3. Immediately immerse in 50:50 mixture of acetone/methanol at room temperature. Allow the coverslips to remain in the acetone/methanol for two minutes 4. Remove the coverslips from the acetone mixture and rinse 2X with PBS 5. Following the above protocol starting step 12 however do not add titron to blocking solution

Discs large (Dlg1) complexes in lymphocyte activation Ramnik Xavier,1,2 Shahrooz Rabizadeh,1. Kazuhiro Ishiguro,2. Niko Andre,2. J. Bernabe Ortiz,2. Heather Wachtel,2. David G. Morris,3. Marco Lopez-Ilasaca,4. Albert C. Shaw,3. Wojciech Swat,5. and Brian Seed,1 1Department of Molecular Biology and 2 Gastrointestinal Unit, Massachusetts General Hospital, Boston, 3 Department of Internal Medicine, Yale University School of Medicine, 4Department of Medicine, Brigham and Womens Hospital, Boston, 5 Department of Pathology and Immunology, Washington University School of Medicine The Journal of Cell Biology, Volume 165, Number 2, July 19, 2004 173178 http://www.jcb.org/cgi/doi/10.1083/jcb.200309044

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