The overall goal of this protocol is to provide a complete three-dimensional view of the capillary distribution in mouse ovarian follicles and show their relationships. The ovary is an essential organ in reproduction and in endocrinology. Count each different developmental stage of follicle and the somatic cells.
In addition, unlike other organs, ovary does not show the repeated branching of the functional unit. The local vascular system may be together with the nervous system are also capable of undergoing major changes in each menstrual cycle in adult females. Applying the passive clarity in the ovary allows us to see the overall distribution and the connection among those cells and follicles in the ovary.
The most of the one key of this measure is that it has the least effect on the ovary, and accounts for the clearing of various markers. Through the reconstruction of the microscopic image we provided a map of the ovary. Some details of the vessels, neurons, and follicles can be observed clearly.
For image analysis, some options of Imaris were used to evaluate the relationships between the ovary and the structures. Prepare profusion solutions according to the article instruction and keep them on ice. Anesthetize the mice and make sure the mouse has no toe pinch response.
Spray ethanol, make an incision on the right atrium, then instantly profuse the left ventricle with 20ml of ice cold PBS at the speed of 10ml per minute to flush out the blood. Then profuse the animal with cold Hydrogel Solution with the same speed. Collect the ovaries as well as the uterus to maintain the integrity of ovarian morphology.
Transfer the separated ovaries to the cold Hydrogel Solution. Under a stereo microscope remove the fatty tissues around the ovary. For better result of tissue fixation, we recommend removing of ovarian bursa.
Keep the ovaries in Hydrogel Solution for three days. After three days, transfer the ovaries into a glass tube and fill the tube with fresh Hydrogel Solution. Stretch the parafilm over the top of the tube and then wrap another parafilm around the neck of the tube.
Ensure there are no bubbles between the parafilm and the Hydrogel Solution. Place the glass tube on a shaker incubator at 37 degrees Centigrade for two hours until gel forms. Carefully remove the excess gel around the ovaries with a paper filter.
Put the ovaries in a 15ml tube and fill the tube with 10ml of clearing solution. Place the tube on a shaker incubator at 37 degrees Centigrade for at least two months. Change the clearing solution every day in the first three days and then weekly until the ovary becomes cleared.
All immunostaining procedures are done at 37 degrees Centigrade on a shaker. First, wash the ovary in PBST for 24 hours. Second incubate ovary in primary antibody diluted by PBST for two days.
Third, wash off the primary antibodies with PBST buffer. Fourth, incubate the ovary with the desired secondary antibody in PBST for two days. Fifth, wash off the secondary antibodies with PBST.
Sixth, transfer the ovary into a refractive index matching solution for RI homogenization. Check the visual transparency, when the ovary has been fully clarified it can be imaged. First, prepare a clean glass bottom dish.
Make a putty cylinder and shape it into a horseshoe shape. Press the outer edge tightly onto glass slide to make a sealed space. The height of the putty should be a little higher than the ovary.
Move the ovary carefully to the center and add mounting solution. Push a dish over the putty and use two putty pieces for fix it. When the ovary is ready to image, Nikon confocal microscope with 16X lens can be selected to image it.
For imaging with confocal microscope, first choose the lens in the Ni-E panel. In the panel acquisition, select the number of channels. First, use Eye Port tab and XY controller joystick to move the tissue to the center of field through checking by microscope eyepieces.
Second, after turning off the shutter light, click live tab to adjust the laser power, HV, and offset for each channel. In the panel Z Intensity Correction adjust acquisition for top layer, click the plus button to define the top, move the lens down lens and set appropriate HV and laser power to the bottom of tissue and add the lowest layer information click To ND to synchronize the setting with the panel and the acquisition. And in acquisition, first set the number of scanning steps.
Click the tabs of Z and large image. Second, go to the scan large image window to define the size of field according to the border of the tissue. Third, back to the ND acquisition to input the size of the scanning fields and choose the overlap within 10 and 50%Then click run Z correction and wait for the imaging process.
First use imagic to open the original NIS file. Click image button, choose color and display channels, then click file, choose image sequences and separately save them as tiff files. Second, use Imaris to open one of the tiff files.
Click edit button and choose the add channels to add the rest. The name and color can be changed in the display adjustment. The thickness of the tissue should also be corrected in image properties.
Third, click edit button and choose Crop 3D to cut the extra part. First, click the filament button and click next. Go to the slice panel, find a tracing vessel and distance, then back to the Surpass, input the static and click next.
Second, adjust the starting and set point threshold. In the camera panel, choose the select, press shift and click on the point to remove the extra part. Third, choose the highest threshold and click next.
Don't select detects points and go next. Some excess part can be removed in edit panel. The color can be edited by clicking the color.
Ovarian vasculature 3D architecture was investigated following immunostaining CD31. The 3D structure can be either observed by slices through other slices or by entirety through volume rendering. CD 31 is staining of adult mouse follicular vasculature was done.
Through the reconstruction of capillaries by the filaments, we could show relationship between the individual follicles and their capillaries. The ovary, like a woman, is always active and interchangeable. However, due to the limitation of traditional histological analysis, it's very hard to describe the relationships among such complex constituents.
The newly developed method, passive clarity may help up us to overcome the optical obstacle, keep the integrity of the whole tissues, and provide the functional clues.
