Experiment 9: Immunofluorescence Localization of Actin and Tubulin



Today, you will visualize proteins in cells via immunofluorescence.  You will stain the cells grown on coverslips with antibodies against tubulin.    This antibody will be allowed to react with the cells and then the unbound antibody washed away.  Then, a secondary antibody that reacts with the first antibody will be used.  This amplifies the signal because more than one secondary antibody molecule can bind to each primary antibody molecule.  Also, the secondary antibody is labeled with a fluorescent dye called Alexa 488 to make it fluorescent.  We will also do a negative control in which we treat a coverslip only with secondary antibody.  This will assure us that the signal we see with both is there because of the reactivity of the first antibody and not the secondary antibody.  Last, we will stain the cells with rhodamine phalloidin, which will bind to F=actin filaments in the cells to visualize the actin cytoskeleton.





A. Attach Cells to Coverslips



1.  Take 4 (18 X 18) coverslips and put them in a P60 dish.  Add 5 ml of HL5 and enough AX2 or NC4A2 cells to create a high density of cells on the coverslip.  You will be imaging at 100x and this will be easier if there are lots of cells on the coverslips.  One simple way to do this is to look at the density of the cells in your growth plate and judge whether you want a higher or lower density on the coverslip.  If lower, triturate the cells and dilute them appropriately into the new dish (ie, if the cells are 2x too dense, add 2.5 ml of triturated cells and 2.5 ml fresh media).  If too low, you can add either add a larger volume to the new dish or spin the cells down to concentrate. The coverslips should be dense with cells, but not overcrowded so they have no room to attach. (~5-8 X 105 cells/ml).


2.  Allow the cells to attach to the coverslips.  If you can do this earlier in the day or the day before, that is preferable. 



B.  Glut/formaldehyde fixation


Your TA will set up several P60 dishes containing 1%/0.1% formaldehyde/glutaldehyde in HL-5 in the hood.  These plates are good up to an hour, but after a certain amount of time will no longer work for fixing, and need to be tossed and made up again. 



It is important to handle coverslips slowly and gently at all stages of the procedure so as not to create shear forces.  The cells are not tightly attached to the coverslips and so are easily removed if you are not gently.  Also, grab the coverslips only by the edge so that you don’t remove cells in the middle.  You can monitor the loss of cells from the coverslips using your benchtop microscope throughout the process.


5. Use forceps to gently transfer your coverslips with the cell side up into the fixing solution. Allow cells to fix 10 minutes in the hood. While waiting, prepare p60 dishes for the next steps.


6.After fixation, transfer the coverslips to a p60 dish containing 0.1% triton X-100 for 5 minutes to dissolve some of the plasma membrane and permeabilize the cells.  Without this step, the antibody molecules will not be able to get into the interior of the cell.


7.  Transfer the coverslips into a P60 dish with 5 ml of Ammonium chloride (NH4Cl). The dissolved ammonium ions will react with the fixative and quench its reactivity. 


Coverslips can be directly added to the parafilm-plus antibody after this step.  It is a good idea to look at your slides at this point to check that your cells are still attached (You don’t want go through staining a coverslip with no cells!).



C.  Immunostaining Cells


Lay out a sheet of parafilm on your bench.  Organize the pattern of coverslips you will use so that you don’t loose track of which are which.  They will not be labeled!


Coverslip1: Negative control-this one will be treated only with Secondary Ab [1/200].  Should be background staining only.  Whatever the secondary antibody happens to react with.

Coverslip2:  This will be stained with a primary mouse anti-tubulin monoclonal  Ab (1/10 dilution) and then secondary goat anti-mouse antibody conjugated to Alexa 488 (1: 200 dilution)  Should stain tubulin

Coverslip3:   This will be stained with  primary mouse anti-tubulin and secondary antibody (1/10 & 1/200 dilutions of antibodies) and rhodamine phalloidin (0.1µg/ml).  Should stain tubulin and actin

Coverslip4:  This will be stained with rhodamine phalloidin (0.1µg/ml) only.  Should stain actin





7.  Onto your sheet of parafilm add:


Coverslip 1:  leave in Petri dish



                                                                                                                  Coverslip 2:  80ul 1/10 dilution of primary antibody in TBST


                                                                                                                  Coverslip3:  80ul 1/10 dilution of primary antibody in TBST


Coverslip 4:  leave in Petri dish


(adjust volumes for coverslips > 12mm square)


8. Onto each droplet, invert one coverslip (cell side down) and allow to stain for 30 minutes. 


It is important at each step to keep track of which side of the coverslip the cells are on so you treat the correct side!!!!


9. Wash the coverslips by moving through a series of 3 small beakers filled with TBST.  Dip your coverslip in each of the beakers, count to five, then move to the next one.  After the last beaker, place the coverslip cell side down onto the next parafilm sheet (below).  Throw these away and replace with fresh TBST when done.


10.  Prepare another parafilm sheet. 



80 ul of secondary antibody diluted 1/200 in TBST



80 ul of secondary antibody diluted 1/200 in TBST


80 ul of secondary antibody diluted 1/200 in TBST +  0.1 ug/ml  of rhodamine phalloidin 



80 ul 0.1 ug/ml rhodamine phalloidin




11. Onto each droplets 1-4, invert the corresponding coverslips from step one (cell side down) and allow to stain for at least 30 min. (In the meantime, you should label your glass slides and add the mounting media to them near the end of the incubation)


12.  Wash 3x as before 3x fresh TBST and then twice in distilled water.  Be careful to do them in the order above, or else change the TBST solutions between coverslips, otherwise the reagents left over from an earlier wash could contaminate your coverslips (for instance, phalloidin in the wash solution could bind to the cells that were not supposed to be treated with phalloidin).


13.  As you remove them from the final wash, blot the coverslip edge on a kimwipe to get rid of as much excess water as possible (you don’t want it to dilute the mounting media) and then invert the coverslip onto 15 µl of mounting media on a labeled slide.  Allow them to dry.  If you see alot of stuff on the dried coverslip, this is salts from the TBST that did not get washed off and crystalized on the surface.  You can clean this off coverslip gently with a Q-tip and water. In order to avoid moving the coverslip and shearing off the cells if you do this, you should first seal the coverslip with nail polish and let it dry for a few minutes. 


14. You can now image the slides with the dry objectives to get a feel for how it worked, and then with the 100x oil objective to take images.  Cells will be visible by phase contrast, but note how different they look after fixation and permeabilization.  The Alexa 488 will imaged with the blue 470 LED and the rhodamine with the green 530 LED.


15.Save your slides in the slide boxes in the –20 freezer. We will use them on Thursday with the confocal microscope.




Questions for consideration:


1.What is the relative localization of microtubules vs actin?  Does the localization of the two probes overlap? Is there any channel cross-talk between the two fluorescent probes (do you see the microtubules faintly in the actin channel or vice-versa)?  Explain!

2.What do the controls tell you?

3.Does the pattern of fluorescence differ from cell to cell? 

4.Is there any background from your secondary antibody?

Reagents/Media:




Formaldehyde/Glutaraldehyde  Fix [(mixed in HL5 just before use]

Formaldehyde 2%  from 16% stock from Polysciences ampule

Glutaraldehyde 0.1%  from 8% stock from Polysciences ampule

Save leftover in 15 ml centrifuge tube in fridge.


Triton X100: 0.1% in [prepared from stock of 20% Triton in H2O]


Prolong Gold mounting media


TBST Buffer [20mM Tris/HCL, pH 7.5,150 mM NaCl, 0.005% Tween 20]


Antibodies:


1o Antibody: use either Koonce’s MAb or anti-alpha tubulin  (12G10) Lab stock [Invitrogen]

Use:1:10 dilution  of any of these  antibody stocks using TBST.


2o Antibody goat anti-mouse conjugated to Alexa 488 [invitrogen]

Use: 1:200 dilution of this antibody stock using TBST


Rhodamine Phalloidin :prepared at 0.1µg/ml in TBST from xx stock



Cell Strain(s):


NC4A2



Other things needed for this experiment:

1.p30 dishes

2.p60 plates

3.18x18 mm coverslips

4.glass slides

5.forceps

6.parafilm

7.adhesive tape




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