Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The overall goal of this procedure is to improve the detection of micro metastases in the mouse lung ex vivo while preserving the inflated lungs architecture. This is accomplished by first perfusing the lung in situ you with PBS to remove the blood, which is the main source of color background in that tissue. The next steps are to inflate the lung by injection of PFA, then pinch off to fix the lung tissue under inflation.

Next, the lung is removed and further fixed in PFA or single lung lobes are embedded for cryosectioning. The final steps are to stain the tissues in Xal solution and visualize the LA zag tumor cells ultimately metastasis on whole lung lobes or in lung tissue sections can be detected down to single cell level with a binocular microscope or a light microscope. The main advantage of this technique over existing methods like the sky standing on its own, is that the detectability of micro metastases is considerably improved due to removal of blood related background.

Begin the protocol by anesthetizing a mouse with an intraperitoneal injection of ketamine, xylazine, and ace promazine in approximately 150 microliters of PBS. When the pain reflexes of the mouse are no longer observed, fix it on the operating table. Back down and slick the hair with 70%ethanol.

Open the skin from the abdomen up to the neck. Pull the skin to the sides or remove it. Carefully open the peritoneum in the thorax to prevent rupture of large vessels like the venina jugulars.

This will improve the efficiency of the perfusion. Now cut the vena cava cardis underneath the liver using a 20 milliliter syringe with a 21 to 24 gauge needle. Slowly inject 10 to 15 milliliters PBS into the right ventricle of the beating heart until the lung is completely white and the heart stops beating.

Then pinch off the blood vessels above the liver and the diaphragm with a vascular clamp. Load a 10 milliliter syringe with 3%paraform aldehyde, and with a 21 to 24 gauge needle inject into the right ventricle. Approximately two milliliters.

Uncover the trachea outside of the thorax and inflate the lung with an injection into the trachea of about three milliliters of aldehyde from outside of the thorax. Then immediately after removing the needle, pinch off the trachea coddled to the puncture with a vascular clamp and wait 10 minutes to allow fixation. Next, transect the blood vessels above the vascular clamp.

Then remove the clamp and inject approximately two milliliters of PBS into the right ventricle to remove excess power formaldehyde. Puncture the lower parts of all the lung lobes with a needle and remove the vascular clamp at the trachea. Now inject three to five milliliters of diluted embedding solution into the trachea coddled to the other injection.

Stop the injection when the paraform hide in the lung lobes is replaced by the solution. Remove the lung carefully by pulling the heart with a forceps and by transecting connective tissue ligaments, blood vessels, and the trachea. Be sure to keep the heart connected to the lungs.

Set aside one lobe for cryo sections and use the remaining lung tissue for whole long staining. Place the lung in a plastic cup with a tight closing lid and fix it with 2%formaldehyde and PBS. Put a piece of gauze on top of the lung to keep it in the solution and let it sit at room temperature for 30 minutes.

After half an hour, remove the fixation solution and wash the tissue thoroughly With PB S3 times, prepare freshly the ready to use xal solution by diluting one milliliter of Xal stock solution, one to 40 in the basic staining solution and add at least 10 milliliters of it into the cup. Sink the lung with a piece of gauze. Secure the lid loosely to allow air exchange and incubate it at 37 degrees Celsius for three to five hours protected from light.

After the incubation, remove the xal solution. Rinse the lungs once with PBS to remove residual xal solution and add at least 10 milliliters of 4%paraform aldehyde in PBS. The tissue is now ready for histology and can be longtime stored in the 4%PFA at four degrees Celsius.

Begin by filling one third of an embedding mold with undiluted poly forze embedding medium. Then load the lung lobe into the mold and continue adding medium until the lobe is completely covered. Carefully avoid making air bubbles now incubate the embedded lobe at four degrees Celsius for 20 minutes.

Next, prepare the embedded lungs for sectioning by slowly freezing them in a mixture of dry ice and isop pentane. If needed, they can now be transferred to a minus 80 degrees Celsius freezer for storage on a cryostat cut seven to 10 micron cryo sections of the lobe mount the sections to commercially available cryos section ready slides Once mounted, immediately incubate the sections in xal staining solution at 37 degrees Celsius for 24 hours in a humidified chamber. The next day, rinse the slides in PBS twice for five minutes per rinse.

Follow this with a brief rinse in distilled water. Now counterstain the slides with nuclear fast red for 10 seconds to get a weak counter stain that makes the xal more dominant and immediately rinse of the counter stain with distilled water. For a darker counter stain, extend the time up to 30 seconds or more.

Mount the slides with immuno mount. The formation of metastases in the done LM eight mouse OS models was reinvestigated. Using the described here representative images of perfused and non perfused lungs of mice simultaneously injected with laxy transduced and nont transduced controlled done and LM eight cells in mice injected with controlled done cells.

Macroscopic and microscopic metastases remained undetectable in non perfused and perfused lungs. But interestingly in mice injected with dun LA Z cells, xgl staining revealed blue micrometastatic foci of single cells or small cell clusters on the surface of non perfused lungs in situ perfusion and fixation of the lungs further improve the detectability of dun la z micro metastases, however, outgrowth to macroscopic foci was not observed in mice injected with control LM eight cells translucent, barely detectable macro metastatic foci larger than 0.1 millimeter in diameter were recognized in non perfused lungs. Perfusion of the lung did not improve the detection of the foci.

However, in mice injected with ate LAC Z cells, multiple X gal stain blue macro metastases were detected on the surface of non perfused organs. Micro metastases were also detected on the surface of non perfused organs. Moreover, perfusion of the lungs further improved the detectability of macro and micro metastases.

Consequently, micro and macro metastases became visible at a higher density and a larger number mainly due to the translucency of the perfused tissue in which foci underneath the organ surface also became visible. An additional histological analysis using cryo sections of lung tissue confirmed the improved detectability of lung metastases in mice injected with LAX Z transduced done and LM eight cells in mice with primary tumors derived from Dun LAX Z cells. Micro metastases or even single cell foci were recognized in lung sections.

This was not observed in mice with primary tumors of the respective control cells. Moreover, macro metastases were also more clearly visible in mice injected with LM eight LA C cells than in animals with the control LM eight cells. After watching this video, you should have a good understanding of how to perform lung perfusion and fixation in ZI two and the subsequent exci staining of the metastasis in the excised lungs.

This technique is a highly sensitive endpoint read out for metastasis distribution in the lung in any kind of cancer study, and can also serve as a benchmark for the improvement of current in vivo imaging techniques such as micro ct PET and MRI used for early detection of metastatic lesions.