RNA Detection

7. Transfer the ovaries in a 20–25μL volume to a nontreated
   clean glass slide with a P200 micropipette using a cut 200μL
   pipette tip


8. With the two tungsten needles separate the ovarioles and flat-
   ten the ovaries as much as possible.


9. Gently lay on top of the ovaries a 2222 mm coverslip, make
   sure that there are no air bubbles trapped between the two glass
   surfaces.


10. Seal the edges of the coverslip with nail polish or with some
    other coverslip sealant.


11. Image with a high magnification, high NA objective that would
    be used for the in vivo imaging and analyze the intensity of the
    specific signal relative to the cytoplasmic background. The
    observed contrast is a good proxy to estimate the in vivo
    performance of the microinjected probes. The following mod-
    ifications of the wash-free FISH protocol will address the dif-
    ferent mechanisms that may underlie the insufficient contrast.

3.6.1 Troubleshooting:
Inefficient Target
Recognition

To make the target segment of the mRNA more available for

hybridization, insert these steps betweensteps 2and 3 of Subhead-

ing3.6 (seeNotes 6and 7 ).

1. Add 1μg/mL Proteinase K to the last IBEX wash and digest
   the specimen for 5 min.


2. Remove the digestion solution and add 500μL IBEX + 0.05%
   SDS preheated to 92C. Incubate for 5 min at 92C.


3. Add 1 mL ice-cold IBEX and chill on ice for 1–2 min. Proceed
   with Subheading3.6,step 3but extend the incubation time to
   60 min.

3.6.2 Troubleshooting:
High Degree of Unspecific
Activation of the Probe
Fluorescence

To test the degree of unspecific activation of the probe make the

following modifications of Subheading3.6 (seeNotes 7and 8 ):

1. Supplement the IBEX used for hybridization at Subheading
   3.6,step 3with 15% EC.


2. After the hybridization (Subheading3.6,step 5) wash the
   specimen with 1 mL IBEX once at 37C and then twice at
   25 C for 310 min.

3.7 Preparation of
Microinjection
Capillaries (Optional)

The aim is to obtain Type B microinjection capillaries with 5–7 mm

long taper and 0.5–1.0μm tips. On a Sutter P-97 micropipette

puller with 33 mm box filament we use the following program:

1. Determine the RAMP temperature of one glass capillary from
   the batch.


2. Enter the following one line program into the puller:

In vivo mRNP Detection by FIT Probes 281