The overall goal of this procedure is to provide an ex vivo observation of the beating of Motr append CLIA located within the mouse brain ventricles. This is accomplished by first harvesting the whole brain and then obtaining thin sections through the lateral ventricles. Next, the slices are incubated in a customized glass bottom plate containing high glucose, medium at body temperature to keep the tissue alive during the experiment.
Finally, the beating of the Celia is recorded using a high resolution differential interference contrast microscope. Ultimately, the structure and protein localization of the Celia can be visualized by immunofluorescence imaging. The main advantage of this technique of our existing method as that it's the first technique to allow classification of the epidermal cells and to three distinct group based on their unique ciliary repeating frequencies and angles.
That's good. The implication of this technique extends toward the therapy of hydrocephalus. As abnormal epidermal CLIA function leads to the accumulation of cerebral spinal fluid in the brain.
Ventricles Demonstrating the procedure will be Mrs. Zaha Alam and Ms.Hannah sat very talented students from our laboratory Begin by cleaning the head of an adult mouse with 70%ethanol. Then starting with the top of the head, use sterile scissors and forceps to pull off the skin and expose the skull.
Next, heal the bone piece by piece, starting from the posterior side and moving towards the anterior side. To remove the skull, elect the whole brain and place it in a 100 millimeter Petri dish of 37 degrees Celsius, high glucose medium. Using a sharp blade, slice the brain on the median sagittal plane to obtain the first 100 to 200 micron section from each half.
Then rinse the sections with 37 degrees Celsius PBS, and immediately place the tissue in 37 degrees Celsius. Eye glucose medium to image the sections, place the brain slices in 30 millimeter glass bottom culture dishes containing one milliliter of high glucose medium, and adjust the enclosed microscope chamber environment to 37 degrees Celsius and 5%carbon dioxide using a 60 x objective oil immersion lens. Next, focus on the cells with regular DIC transmitted light.
Then following the direction of the DMEM bubble movement as a guide to the location of the motil append clia. Use the DIC filter to select an area containing healthy cells with motil CLIA in the brains lateral ventricle. Upon identification of the append clia, adjust the light and focus to obtain a satisfactory image.
Next, set the live imaging parameters in the imaging software. For example here, 24 bit images are being acquired with the camera bending set to one times one combined with the 60 x objective and a five to 10 millisecond exposure time. Open the microscope aperture to the optimal level for a minimal exposure time and collect the DIC images.
Observe the live images streamed to the camera to provide fast and immediate image acquisition without delay. The speed of silly beating can then be calculated based on the requirement of the minimal exposure times for obtaining a sufficient image contrast to record calcium signaling. After slicing the brain, briefly rinse the tissue in PBS and immediately incubate the sections in 20 micrograms per milliliter of freshly prepared lu O2 in a glass bottom plate.
After 30 minutes at 37 degrees Celsius, record the calcium oscillation at a capture rate of five milliseconds for a minimum of one second. First at an excitation wavelength of 488 nanometers, then at an emission wavelength of 515 nanometers. To visualize the cell markers of interest by immunofluorescence microscopy, fix the samples in 4%para formaldehyde and 2%sucrose in PBS for 10 minutes.
Wash the tissue three times in PBS for five minutes each wash. Then incubate the brain slices in 0.1%Triton X for five minutes. After rinsing the samples as just demonstrated, incubate them with mouse anti acetylated alpha tubulin.
After another PBS wash, incubate the samples in fluorescein anti-US IgG for one hour at room temperature. Finally counterstain the sections with DPI for five minutes and immediately image the cells with the minimum possible exposure time. The presence of a penal CLIA is confirmed with a ciliary marker acetylated alpha tubulin with DPI counter staining To identify the nuclei analysis of the stream of a penal CLIA motility is accomplished by counting the beating frequency and angle pattern of the moving cells.
The CLIA can then be divided into one of three groups based on these data. Importantly, exposure of the CLIA to 0.25%Ethanol significantly represses the CLIA beating frequency irrespective of their type. Further bending of the CLIA triggers a cilium dependent intracellular calcium signaling, which allows measurement of the intracellular calcium signal within the brain ventricles Once mastered, this technique can be completed in one hour if it's performed properly.
While attempting this procedure, it's important to remember to handle the brain tissue gently and efficiently, so as to not destroy the brain ventricles After its development. This technique paved the way for researchers in the field of neuroscience to explore the function of append CLIA and live mouse brain tissue.
