This protocol shows the appropriate methodology for heart and brain tissue slicing and staining. It also provides guidelines for establishing lighting and camera setups and photography techniques for image acquisition. These methods are particularly useful for performing measurements in image planimetric analysis in rodent tissues, and can be of value for general scientific macrophotography.
To begin, detach the heart cannula from the syringe filled with Krebs-Henseleit solution and connected to a syringe filled with warm Krebs-Henseleit solution containing 0.1%methylene blue. Perfuse the rat heart with four milliliters of the methylene blue solution at a rate of four milliliters per minute. After disconnecting the cannula from the syringe and removing the heart from the cannula, remove the excess methylene blue by gently rolling the heart on tissue paper.
After removing the excess methylene blue, loosen the ligature around the coronary artery by opening the hemostatic forceps and removing the plastic tubing around the surgical suture. Next, place the stained rat heart in a stainless steel matrix for slicing. Cut the ventricles into two-millimeter-thick slices, aiming for six to seven slices from one adult rat heart.
After cutting, transfer the slices to a 15-milliliter plastic tube. Then, add five milliliters of 1%triphenyl tetrazolium chloride dissolved in PBS to the tube with the heart slices and incubate for 10 minutes in a water bath at 37 degrees Celsius with gentle mixing after five minutes. After incubation, wash the heart slices at least two to three times with PBS and prepare for image capture.
After removing the brain, including the brain stem, from the skull, place the brain with its ventral side up in the brain matrix. Depending on the weight of the animals, choose the correct size of the brain stainless steel matrix. Using blades, restrict the frontal and caudal parts of the brain.
Then, put the blades partially into the channels between the first and the last blades. When all the blades are inserted and arranged in parallel, press all the blades down with the palm at the same time to cut the brain into two-millimeter coronal slices. Then, grasp the blades firmly along the sides with two fingers and remove them together with the sliced brain from the matrix.
Arrange the brain slices one by one in a tray, ensuring that the interior surface of each slice is always facing up. Next, pour warm 1%triphenyl tetrazolium chloride solution and PBS onto the brain slices and incubate for eight minutes at 37 degrees Celsius in the dark. After incubation, transfer the brain slices into a blue plastic tray to capture images.
Arrange the brain slices in sequential order from the frontal to the caudal part and use a scalpel to separate the hemispheres in the sagittal plane. Photograph the tissue slices immediately after staining. Set up the camera of choice with a charged battery, memory card, and attached lens on a stand.
Depending on the available light sources, select the appropriate white balance settings or perform color temperature calibration according to the instructions in the camera manual. Immerse the heart slices completely in a container with PBS. Arrange the brain slices in a dry tray without PBS or other liquids.
Place the container with slices under the camera with the macro lens. Ensure that all slices fully fit in the field of view and are on the same plane. Switch the camera to fully manual mode, set the ISO 100, and aperture to F10.
Adjust the shutter speed for optimal image exposure and ensure that the camera focal plane is parallel to the surface where the sample is place. After capturing the sample number, rotate the circular polarizing filter until reflections disappear, and image the tissue slice using a remote trigger to prevent a camera shake when the shutter is released, then rotate the slices and capture images from the other side. This photograph of a methylene blue and triphenyl tetrazolium chloride-stained heart slice after myocardial infarction contains enough detail and color information for further planimetric analysis of infarct size.
Freezing of heart tissue for 24 hours affects the integrity and reduces mitochondrial function. Thus, triphenyl tetrazolium chloride staining of the heart is not red but pale pink, and the border between the necrotic and viable tissues is blurred. Out of the two methods compared for reducing reflections in the specimens, immersion is the most efficient method and produces detailed images with good contrast.
The polarizing filter is also effective, however, it slightly reduces the resolution and microcontrast of the image. An image of a heart slice without immersion or filter contains many reflections and is not suitable for further analysis. In the planimetric analysis, the unaffected side of the brain should be compared with the stroke-affected side.
Manual camera settings ensure optimal exposure and white balance of all images, allowing uniform analysis. Examples of overexposed and underexposed images of heart slices are shown here. Furthermore, incorrect white balance settings result in a shift to blue or yellow and magenta or green cast in the image.
Scientific macrophotography requires controlled lighting and an appropriate imaging setup. The standardized methodology ensures high-quality, detailed digital images. This method is applicable to all experimental small animal organ photography.
