The goal of the following procedure is to use multi-spectral interrogation of multiplex cellular compositions or mimic to determine the contributions of various bone marrow resident populations to tumors stroma development. This is accomplished by first transplanting a transgenic GFP labeled recipient mouse with RFP expressing bone marrow stem cells. In the second step, the tumor cells of interest are injected into the same recipient and then after engraftment, the excise tumor is sectioned, embedded in paraffin and labeled with immunofluorescent antibodies.
Next, a spectral library of all the wavelengths used for acquiring images of the tumor sections is created once the spectral library is complete. The slides are imaged in the final step, data analysis is performed to allow the identification and association of the staining patterns of the cellular markers of interest. Ultimately, novel cellular entities or phenotypes can be identified based on the multiplex labeling of extracellular and cellular markers to better understand the composition of complex tissues such as the tumor microenvironment.
The main advantage of this technique over standard immunofluorescence techniques is the multi-spectral analysis allows you to multiplex seven or more fluoro fours on the same slide, allowing for a much more precise and detailed interrogation of the cellular composition of the tumor microenvironment or a regenerative tissue. Though this method can provide insight to the origin and composition of the tumor microenvironment, it can be applied to other systems such as regenerative medicine, developmental biology, and immune biology. Demonstrating this procedure along with Dr.Spa is Chris Booth, a research assistant in my laboratory Due to IU Cook restriction at this institution, we will discuss but not show any direct mouse manipulations on mice.
These bone marrow procedures and our tumor engraftment techniques can be found in our prior work, such as kid and others stem cells. 2009 to begin after isolating the bone marrow cells from the mirroring hind limbs, resus suspend the cells at 2 million cells per 100 microliters of PBS per mouse. Use a heat lamp to dilate vessels of an irradiated recipient GFP mouse and place the mouse in a tail vein injection restraint.
Using a 29 gauge needle bevel side up systemically inject the cell suspensions into the tail or retroorbital vein. Use sterile gauze to apply light pressure to the tail post-injection. Inject one mouse with 100 microliters PBS only as engraftment control on the day of injection.
Re suspend the tumor cells of interest in PBS at 1 million cells per 100 microliters. After sterilizing the abdomen with 70%ethanol. Use one hand to restrain the mouse by the scruff of the neck and the tail, and then with the other hand, hold the needle bevel side up and inject the tumor cells into the interperitoneal cavity of the recipient mouse.
After 15 to 30 days, euthanize the mice and excise the tumor from its abdomen. Then fix the tumors in 10%formin for 24 hours. Then section the fixed tissue into five to 10 micrometer slices for subsequent staining procedures.
After fixing the tissue sections in paraffin, bake the samples for one hour at 56 degrees Celsius. Then wash the slides according to the following sequence After the last ethanol wash, rinse the slides in water for two minutes. Slowly submerging and raising the staining rack in and out of the water 10 times.
Next place sodium citrate buffer in the microwave to heat. After boiling the slides for 20 minutes in sodium citrate buffer, let the samples cool in the buffer for 30 minutes. Then wash with the ionized water for another five minutes.
Then tap off the slide to let any big drops of water roll off, take caution to not dry the tissue section out. Use a hydrophobic barrier pen to mark the sections around the tumor sections and then immediately place blocking buffer on the sample to prevent the tissue section from drying out. After an hour, incubate the slides with the primary antibody of interest in a moisture chamber box on a slow rotating shaker at four degrees overnight.
The next morning, wash the slides two times in PBST for 10 minutes each time with gentle shaking while the sections are being washed. Dilute the secondary antibodies at a one to 1000 antibody to blocking buffer ratio. Then incubate the sections in the appropriate secondary antibodies in the moisture chamber box covered with aluminum foil on a slow rotating shaker at room temperature After two hours.
Wash the slides two times in PBST for five minutes with gentle shaking and protect it from light. Then stain the dappy only slide and the analysis slides with DPI for one minute, keeping the slides covered during the incubation. Placing a cover slip over each sample, seal the edges of each cover slip with nail hardener.
Now to perform the multispectral imaging, use an oil objective to take an initial image of the analysis slide to obtain the proper exposure times for each of the spectral ranges in the spectral library. For example, this table outline suggested spectral library protocols based on the number of floor fours used in the experiment. Then image the unstained slide for the background slide in the spectral library for this experiment.
Next image, the unstained slide to achieve the background image for the spectral library. Now image each control single floor four stained slide, saving the images under the name of the secondary antibody used for each slide. Once Suspectable library is complete, collect images of the regions of interest on the analysis slide to analyze images of the tumor tissues.
First, import a representative assortment of the acquired nuance images using inform software. Then open the safe spectral library as directed by the program. Next, identify the tissue regions of interest to be analyzed.
Identify individual cells based on DAPI staining. The cytoplasm will be identified automatically based on the shape of the nucleus. Now pick the fluorescent intensity readout of choice for each fluorescent parameter, and then match the acquired images, letting the software process the data using the proposed algorithm.
When the image processing is finished, merge the resulting files. Next, using this multi-spectral imaging technique to analyze and graph tumors in this transgenic bone marrow transplant mouse model, stromal tumor components that are of bone marrow origin were discerned by flow cytometry three weeks after bone marrow transplant. Then six weeks following the initial injection sections from the unlabeled injected ovarian tumors were analyzed after creating a spectral library with single color control slides, images of the ovarian tumor section stained with GFP DPI and two other markers were analyzed.
The addition of more markers is possible. For example, here, the spectral library and accompanying image of a tumor section with six markers plus a nuclear stain are shown using the informed software. The tissue regions of interest within tumor section are classifiable as shown in the representative images shown here.
For example, this image demonstrates the classification of tissue segments followed by the segmentation of the cells. Based on nuclear stain dpi. Quantification of the fluorescent labels can then be measured in the nucleus and cytoplasm of each cell and exported for further analysis.
The tissue segmentation algorithm is capable of further identifying tissue subtypes based on tissue architecture and not solely on fluorochrome identification, providing a more robust analytical tool to classify the staining into distinct tissue regions. In this image, the computer was trained to identify five sub regions based on the architectural patterns within the tissue. The background is defined by blank space.
The hemolytic tumor is defined by the abundance of red blood cells within the tissue. The tumor is defined by the densely populated cells. The fat is defined by the large symmetrical empty cellular compartments and the muscles defined by a striated pattern.
Using this method, a double positive cell population can be analyzed based on a determined fluorescent intensity threshold. For example, for GFP positive and alpha smooth muscle actin positive cells, as shown here for additional spectral detection beyond four colors, please see the linked tips and tricks documents for multiplexing five or more probes. While attempting this procedure, it's important to remember that the quality of the images relies highly upon the proper single color control slides used.
Following this procedure, other methods like fluorescence in situ hybridization can be performed in order to answer additional questions such as cellular compartments and tumor microenvironment tissue. After watching this video, you should have a good understanding of our mimic technique, the multi-spectral interrogation of multicellular components, and when completing this, you should have a thorough understanding of how to identify multiple cellular targets on one slide. How to tally Unix those cellular targets and have a good understanding of the evolution of tumor stroma or regenerative tissue, or your tissue of interest.
