Staining formalin in PBS for at least 30 minutes.Reagents

Staining Protocol to visualise Cytoskeleton StructureIn order to view the cytoskeleton structure (in this case, the actin cytoskeleton), HeLa cells will be fixed onto coverslips, transferred to a microscope slides and then stained to be viewed under a DSD confocal microscope which will outline structures such as stress fibres, lamellipodia and filopodia through fluorescence. The nucleus, as well as the focal adhesions, should also be visible.HeLa is part of an immortal cell line that is the most common and widely used cell type in scientific research due to being very durable and prolific. The cell line was derived from cervical cancer cells taken on February 8 1951, from Henrietta Lacks, who died of her cancer on October 4, 1951 (Capes-Davis et al, 2010; Rahbari et al, 2009; Syverton & Scherer, 1952).Tissue culture procedureThe cells are seeded onto sterile glass coverslips in a 6 well plate at 1×105 per well (or optimum for cell line.). The cells are then allowed to grow for 24-48 hours under the appropriate cell culture conditions. After this, it is fixed in 4% formalin in PBS for at least 30 minutes.Reagents and pre-practical protocolAfter fixation has taken place, a permeabilisation/blocking Buffer is used to permeabilise and block the cells. The buffer used is 0.5% Triton-X 100 and 2% Goat serum Albumin in phosphate buffered saline, and this is done for 30 minutes. The permeabilisation/blocking buffer is then removed and a 200µl measure of primary antibody anti paxillin made up in a 1:100 dilution ratio with permeabilisation/blocking buffer is added to each coverslip.The cells are then left overnight at a temperature of 4°C. After this, the antibodies are then removed and the cells washed in PBS.1ml of FITC-Phalloidin/DAPI/ Alexa Fluor 568 goat anti-rabbit combined stain:• 1µl Alexa Fluor 568 goat anti-rabbit• 979µl DAPI stock solution (DAPI stain is made up from a working concentration of 250ng/ml)• 20µl FITC-phalloidin stock solution (FITC-Phalloidin stock solution is 0.05mg/ml)Class Practical Protocol1. The cells are washed twice for one minute each with a measure of 2ml PBS using a Pasteur pipette.2. Then an approximate of 500µl FITC-Phalloidin/DAPI/ Alexa Fluor 568 goat anti-rabbit combined stain is added onto each coverslip. This is then left for one hour in an area which is dark while at room temperature (e.g. simply put an opaque box on top). The cells must be kept in the dark for the remaining of the protocol.3. Each of the microscope slides that are to be used are labelled with the group number, date of the practical, as well as the cell line and the name of the primary antibody used.4. The cells are then washed once again with a measure of 2ml of PBS twice for two minutes each.5. The coverslips are then mounted using soft set Vecta Shield (This is an anti-fade mountant) and then fastened to the microscope slides using nail varnish. The stained slides are then stored in the dark (Once again, this can be as simple as under an opaque box).6. The final step is to simply use a DSD confocal microscope view the microscope slides.Different cellular structures will be observed with different colours according to which of the three stain we have used that they have taken up. • The cytoskeletons observed should be green, due to the use of FITC-Phalloidin.• Focal adhesions will show up as red, and this is the result of Alexa Fluor 568.• Finally, the nucleus and DNA material will show up as blue due to DAPI.RESULTSFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6Figures 1, 2 and 3, 4 are used here to compare side by side the focal adhesions and actin cytoskeleton fluorescence via staining and confocal microscopy. As you can see in these comparisons, the red indicates focal adhesions due to Alexa Fluor 568 stain. From our results, it suggests that the focal adhesions are more prominent in the leading area of the cell.   The green indicates the actin filament cytoskeleton (due to FITC-Phalloidin) which is dispersed throughout the cell but is concentrated in the cortex. Apart from figure 1 and 2, the actin cytoskeleton structures are visible in all the other figures showing stress fibres, filopodia and lamellipodia. We can see very well from figure 5 that stress fibres (green) terminate in focal adhesions (the red). Figure 3 is very good at showing the filopodia projections and figure 2 highlights the lamellipodia. Figure 6 shows multiple cells with the different actin filament structures. In all 6 figures, we can see the nucleus very clearly and this is due to the DAPI stain.CONCLUSIONThe recent advances in various microscopy techniques and methodologies enable cell biologists to observe and visualise complex cellular processes. Imaging technologies allow us to view the cytoskeleton structures and advance our understanding of its functions in the cell.  Fluorescent labelling and visualisation offer high sensitivity, specificity and the capability for quantification.  This is particularly relevant for actin filaments as the dynamics and assemblies of the proteins are very complex.  The actin cytoskeleton is usually visualised with chemical tools such as fluorescently-labelled phalloidin. This method, however, has limited application in living cells. (Mckayed & Simpson, 2013)

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