This technique allows for not only localization, but also quantitation of candidate mRNAs in the individual oocytes and embryos. Compared with other assays, including PCR, completion of the technique results in reproducible data with low variation. Demonstrating the procedure will be Kelsey Timme, a graduate student in my laboratory.
Begin this procedure with the preparation of the required material and female mice at five to eight weeks of age as described in the text protocol. Clean the mouse using 70%ethanol. Expose the abdominal cavity and visualize the female reproductive tract.
Hold the ovary with forceps and remove the uterine ligaments and excess adipose tissue from around the ovary. Cut the oviduct from the uterus. Then, place the ovary oviduct pair in warm holding medium in a 35 millimeter dish.
Remove the ovary and any surrounding adipose tissue. Tear the swollen ampullae of the oviduct using a half inch 27 gauge needle. Push the oviduct at the site of the tear and cumulus cell oocyte complexes will be expelled.
Using a mouth pipette to transfer the ovulated oocytes to the 100 microliter drop containing holding medium with hyaluronidase. To dislodge cumulus cells, pipette the metaphase two oocyte cumulus cell complexes up and down in the hyaluronidase containing holding medium with the mouth pipette. Once they are devoid of cumulus cells, use the mouth pipette to transfer each oocyte to a wash drop containing only holding medium.
Repeat this for each wash droplet. Do not transfer fragmented or transparent oocytes. To perform SM-FISH staining, first fix oocytes in an individual well of a six well plate containing 500 microliters of fixation buffer.
Submerge 20 oocytes or less in the well. Incubate the oocytes in fixation buffer for 20 minutes at room temperature. After washing the oocytes as described in the text protocol, incubate oocytes in permeablization buffer for 30 minutes at room temperature.
Following another wash, transfer the oocytes to 80 microliters of pre-diluted protease three buffer for 30 minutes at room temperature. Dilute the warmed probed sets for Nanog, Pou5f1, and DapB one to 50 in probe diluent. Incubate oocytes in 80 microliters of the transcript specific probe for two hours at 40 degrees Celsius.
When oocytes are in the probe and the AMP buffers, they tend to float. It is essential that you submerge the oocytes by gently pushing them into the buffer. Transfer the oocytes to 500 microliters of wash buffer and incubate for 10 minutes at room temperature.
Now, incubate oocytes sequentially in amplification buffers. First, incubate oocytes in 80 microliters of AMP1 for 30 minutes at 40 degrees Celsius. Then transfer the oocytes to 500 microliters of wash buffer for 10 minutes at room temperature.
Next, incubate oocytes in 80 microliters of AMP2 for 15 minutes at 40 degrees Celsius. After washing the oocytes as before, incubate the oocytes in 80 microliters of AMP3 for 30 minutes at 40 degrees Celsius followed by a final wash. Finally, add oocytes to 80 microliters of AMP4FL for 15 minutes at 40 degrees Celsius.
Pipette 12 microliters of anti-fade mounting medium onto the center of a slide without adding bubbles to the reagent. Transfer oocytes with as little wash buffer as possible into the mounting medium. Finally, apply a cover slip.
Image the three dimensional oocytes using Z-step confocal microscopy and save the images as described in the text protocol. Drag ND2 files into Fiji and choose Hyperstack. Click the Image tab, select Color, and click Split Channels to separate the fluorescent channels of the ND2 file.
Generate individual TIFF files for each Z slice of the oocytes in each fluorescent channel. To do so, click the Image tab, select Stacks, and click Stack to Images. Then, click the Image tab, select Type, and click RGB Color to convert each Z slice to an individual RGB color image.
Save each converted image as a TIFF file. Place images from a single oocyte for each fluorescent channel in a new folder to avoid confusion during stitching. After normalizing the files as described in the text protocol, click the Plugins tab, select Stitching, and click on Grid Collection.
Select Sequential Images from the dropdown menu and click Okay. Browse the directory and select the folder containing all of the Z slice images for an individual oocyte at one wavelength. Click Okay.
Move the slider at the bottom of the stitched image to the appropriate color channel for the wavelength used. Create the final RGB stitched image by clicking Image, selecting Type, and clicking RGB Color. Convert the stitched image to a 32-bit maximum projected picture.
To do so, click Image, select Type, and click 32-bit. Save this image as a new TIFF file. Open the 32-bit stitched image in a spot finding and tracking program.
Select the Localize dropdown and click Localize, which will calculate the number of spots found in the image. Shown here is the quantification of mRNAs using the spot finder and tracking. The blue arrow points to a positive signal above threshold.
The white arrow shows a fluorescent spot below the threshold and therefore not counted. The mRNA counts were subsequently analyzed using a standard data analysis tool. Representative images of the middle Z series image are shown for Pouf51 and Nanog.
DAPI staining of chromosomes aligned on the metaphase two spindle is shown in white. The data collected in this protocol showed 775 Pouf51 transcripts and 113 Nanog transcripts in metaphase two oocytes. Importantly, there were no spots detected in oocytes that are labeled with DapB probe, which served as the negative control.
When using non-adherent cells, it is important to empirically identify the best dilution of protease buffer from the SM-FISH kit. mRNA detection was tested using a titration curve of undiluted and several diluted protease buffer concentrations in 1xPBS. Protease dilution used in this protocol was one to eight as it showed the lowest variation in the average fluorescent expression of Pouf51 mRNA in metaphase two oocytes.
Concurrent immuno-fluorescence of a target protein can be performed in order to co-localize the mRNA with an RNA binding protein. Co-localization of the mRNAs with the RNA binding proteins allows investigators to determine mechanisms that regulate mRNA storage, translation, and degradation within an oocyte or embryo.
