Biology Reference
In-Depth Information
troubleshooting. All the proteins involved have to be tagged for visualization in the
TIRF microscope and rigorously purified to avoid artifacts due to contaminating
proteins.
8.1
PROTOCOLS
8.1.1
Preparation of coverslips and slides
For fluorescence microscopy-based
in vitro
experiments, small glass sample cham-
bers are assembled from microscope slides and coverslips. It is crucial that all glass-
ware is thoroughly cleaned, as the major contaminants for TIRF microscopy, which
tend to be organic in nature, exhibit autofluorescence. Cleaning can be done in sev-
eral ways. If one has access to a plasma cleaner, then this is perhaps the easiest
method. However, there are also inexpensive chemical alternatives. We have
employed two methods in the lab that successfully remove contaminants.
8.1.1.1
Chemical cleaning of slides and coverslips
One method is to soak the slides and coverslips overnight at room temperature in a
saturated KOH solution dissolved in ethanol (
16.5 g of KOH pellets per 100 ml of
ethanol,
note
: this produces an extremely basic solution, and hence care should be
employed as the solution is highly corrosive and the ethanol is highly flammable).
For safety, use the smallest volume required to clean the glass; we use approximately
50 ml to prepare a set of slides and coverslips. The next morning, remove the slides
and rinse thoroughly in Milli-Qwater six times. The slides and coverslips can then be
stored in fresh Milli-Q water for up to 1 week, ensuring that they are protected from
dust and other particulate matter. Use slide and coverslip holders made from glass or
ceramic material during the overnight soaking step so as to provide efficient
cleaning.
8.1.1.2
Sonication of slides and coverslips
The second cleaning method that we have employed is based on sonication. Rest the
slides and/or coverslips in an appropriate holder and place them in a clean beaker
(100 or 200 ml depending upon the size of the holder). Add a solution of pure iso-
propanol, 0.2
m filtered, until it passes the coverslips/slides by approximately 1 cm
(see
Fig. 8.1
). Next, sonicate the mixture for 20 min. After sonication, wash the glass
in Milli-Q water three times. Make up a 1 M solution of KOH, 0.2
m
m filtered, and
add it to the beaker, again making sure that there is at least a 1 cm clearance above the
glass, and repeat the sonication for further 20 min. Remove the KOH and wash fur-
ther three times in Milli-Q water. Store the slides and coverslips in fresh Milli-Q and
an appropriate container until use (do not keep them longer than a week). Using this
method, a batch of coverslips can be prepared quickly in less than 2 h. The results are
comparable to the standard chemical cleaning as outlined above.
m
