The overall goal of this protocol is to quantify protein levels in situ in mouse preimplantation embryos. So this method allows us to perform high-throughput analysis of confocal microscopy images with single-cell resolution in a semi-automated way. This method can be used to quantify protein concentrations on confocal images where there is high nuclear density and when one wants to study heterogeneities within cell populations.
To begin, use an open flame on a glass pasteur pipette to draw a capillary end at the tip. Gently rub the far end of the pipette against the capillary, to break it while creating a blunt end. After sacrificing a pregnant female mouse on the desired embryonic day of development, expose the abdominal viscera according to the text protocol and locate the uterus.
Hold the cervical end, cut through the cervix, and gently pull up to stretch both uterine horns. Trim the fat from it, taking care not to pierce the uterine wall. Cut above the oviducts, below the ovaries, to release the entire uterus, and place on a petri dish.
Next, cover the uterus with PBS, and separate both uterine horns by cutting through the proximal end on both sides of the cervix. Then separate each oviduct from the uterus by cutting below the isthmus that connects them. Under a dissection microscope, use a one mL syringe with a hypodermic needle to flush the blastocysts out of the uterus and into manipulation medium, by forcing 0.5-1 mL of medium through each horn from the cervical opening.
Allow up to one to two minutes for blastocysts to sink to the bottom of the dish after flushing. Then locate the blastocysts, and using the mouth-controlled glass pasteur pipette with a capillary end, collect the blastocysts and transfer to a fresh drop of medium. After rinsing the blastocysts, remove the zona pellucida by using Acidic Tyrode's Solution to briefly wash the embryos.
As soon as the zona is no longer visible, transfer the blastocysts back to manipulation medium. Wash the blastocysts in room temperature PBS, and fix them by transferring to 4%PFA in PBS for 10 minutes at room temperature. After fixation, transfer the blastocysts back to PBS for storage at four degrees Celsius.
After carrying out imunoflourescence according to the text protocol, use a fine mouth pipette to make microdrops of PBS or nuclear stain solution on the glass surface of a 35mm glass-bottom dish, and cover them with mineral oil. Place the embryos in the microdrops and arrange in a consistent manner, preferably with the ICM cavity access parallel to the glass surface, then set up the dish on the microscope holder. To carry out confocal imagining, use the lowest laser power that provides a strong signal-to-noise ratio without bleaching the fluorophores.
Adjust the gain and off set to obtain the widest dynamic range without overexposing the sample. Capturing most of or the entire grayscale range will facilitate the detection of small differences in intensity between images. Follow the additional details outlined in the text protocol to image embryos throughout the entire Z axis.
Follow the graphic user interface of the MATLAB-based segmentation tool Modular Interactive Nuclear Segmentation, or MINS, to load a confocal image or an entire Z stack of raw data generated by the microscope. To view the outcome of each step, click the corresponding View"button. A yellow tag above the button indicates operation in process.
A green tag indicates operation completed. Assess the outcome of the detection step before proceeding to segmentation. If it is not satisfactory, modify the detection parameters and rerun it.
From the Batch Mode"run menu, click Add Files"and load all files to be processed at once. Click Start Batch Mode Run"and allow time for the software to process the files. Segmentation output will be saved in the same directory as the original files.
Identify false positives such as apoptotic vesicles or other elements that are not intact nuclei but which may have been identified as such by MINS. Delete the corresponding records for false positives from the starstatistics. csv file.
Preserve the original starstatistics. csv file for future reference and edit only a copy of it. If a nucleus has been over segmented and presented as two or more nuclei, either merge the records by averaging their intensity level, or keep one of the records for that cell and discard the rest.
To resolve under segmentation, identify events where MINS has failed to detect the border between two or more nuclei and segmented them as a single one, or where it has failed to detect a nucleus altogether. Use ImageJ to measure the average gray level for each channel in the under or unsegmented cells. Next, find a medial section of the under or unsegmented nucleus on the DNA channel, using the freehand selection tool, outline the perimeter of the under or unsegmented nucleus.
Then, press Control M"or go to the Analyze"menu and select Measure"This will record the mean gray value for the area outlined and will display it on a new window. Using the same outlined area just selected in the DNA channel, repeat the measurement of each of the fluorescence channels of interest. The results will be upended to the previous one on the measurements results window.
Then use the measurements obtained to replace the erroneous records in the starstatistics. csv file. If the nucleus has been undersegmented duplicate its row, assign different cell IDs to each new cell, and introduce the values obtained in ImageJ under the corresponding column.
In this case, both cells will share spacial coordinates. When mitotic nuclei are to be considered separately in the analysis, manually score them and add the information to the data file. Refer to the text protocol for additional image processing instructions.
This figure shows examples of intact blastocysts at different stages with an expanded cavity. Shown here are examples of good antibodies for a number of nuclear proteins, including CDX2, GATA4, GATA6, NANOG, and OCT4. This sample was labeled with the cytoplasmic protein DAB2 that gives a high signal-to-noise ratio.
In this panel, examples of a bad staining for GATA4 with a low signal-to-noise ratio are shown, where the samples were only fixed for 10 minutes. This particular anti-GATA4 antibody requires overnight fixation to provide a strong signal. These embryos were imaged with a 40x oil immersion objective with an NA of 1.30 and a 0.21 mm working distance The middle panels show magnifications of the ICMs or individual nuclei and the border between them can be distinguished.
The bottom panels illustrate how the more advanced the embryo, the higher the nuclear density, and thus the greater the chance of segmentation errors. These images show errors that MINS can commit, such as detecting apoptotic nuclei as live cells, over segmentation, or under segmentation. This sequence of Z slices reveals an under segmentation event, where two cells have been identified as one.
Once mastered, this protocol can be carried out in three to four days from beginning to end, with a time burden of two to four hours per day. While performing this procedure, it is important to remember to be very consistent with experiment conditions, that is immunofluorescent protocols and imaging parameters. After its development, this technique paved the way for researchers in the field of mouse developmental biology to perform in situ quantitative analysis of gene and protein expression in single cells.
After watching this video, you shall have a good understanding of how to acquire image data that is appropriate for quantitative in situ expression analysis.
