- Since PFA and formaldehyde covalently cross-link protein
molecules to RNA and also break the sugar phosphate back-
bone of nucleic acids, prolonged fixation will result in greatly
reduced FISH quality. - Since the purpose of the wash-free FISH analysis is to keep the
RNA in its native state, those steps of a classical FISH protocol
that make the RNA more available for hybridization (e.g.,
limited Proteinase K digestion and denaturation of RNA sec-
ondary structures by high temperature) are omitted. - These modifications will reduce the likelihood of unspecific
binding. Optionally the hybridization temperature may be
increased up to the melting temperature of the probes. - If neither of the modifications described in Subheadings3.6.1
and3.6.2improve the signal to background ratio, the likely
cause of the poor contrast is the insufficient activation of the
probe fluorescence by duplex formation (low in situ respon-
siveness). While the inefficient target recognition of the probe
inevitably requires that a different segment of the RNA is
targeted, insufficient or unspecific activation of the probe fluo-
rescence may be solved by moving the TO dye to another
position within the same sequence. - In case of a gas operated injector, load the microinjection
needle from the back with an Eppendorf capillary loading tip.
Make sure that the solution reaches the very tip of the capillary
before inserting it into the holder. Use gravity and/or firm
shaking of the capillary to increase the speed of this process.
When using an oil based injector, plunge the tip of the injector-
mounted needle into a drop of the probe solution placed onto a
coverslip under the injection microscope. Apply negative pres-
sure to load the tip from the front. - Plunging the tip into any liquid drops on a glass surface
requires special care to prevent damaging the needle. Turn on
the transmission light of the microscope and navigate the tip
into the light cone while keeping a 1–2 cm distance from the
glass. Once the light gleams on the needle, gently lower it until
it touches the surface of the liquid. Then switch to the eyepiece
of the microscope and slowly move the needle in the lateral
directions (x and y) until you find the tip. Usually, it appears
rather unfocused, mostly just as a shadow. Focus to it by using
the focus control of the microscope. Then by coadjusting the
axial position of the micromanipulator and the microscope
focus, the tip can be lowered slowly until it reaches the glass
surface. When the tip is seen bending elevate its position
slightly to prevent any damage to occur. - Sometimes the tip of the needle is sealed—especially if it was
self-made—or gets clogged. If no liquid bubbles appear despite
In vivo mRNP Detection by FIT Probes 285