The overall goal of this procedure is to label and visualize M-T-D-N-A replication in cultured neurons. This is accomplished by first incubating neurons with the thymidine analog EDU for two to 24 hours. The second step of the procedure is to label the EDU that contains an alkin with the fluorescent orgon green 4 88 azide through a copper catalyzed covalent reaction.
The final step of the procedure is to amplify the orgon green signal with aramide signal amplification step in order to visualize the EDU that was incorporated in newly synthesized MT DNA. Ultimately, results can be obtained that show MT DNA replication in specific subcellular compartments of neurons through the combination of clicka EDU chemistry and subsequent tear mind signal amplification. The main advantage of this technique over existing methods like BRDU labeling, is that EDU labeling is easier and milder, allowing for additional immunofluorescent staining of neuronal markers.
This method can answer key questions in the regulation of mitochondria number, such as how neurons meet changing energy loads in their subcellular compartments, including cell bodies, axons, dendrites, and synaptic terminal. So this method can provide insight into the mechanisms underlying the pathogenesis of multiple mitochondrial based neurologic diseases, including neuropathy and neurodegeneration. But importantly, it can also be applied to other systems where changes in mitochondrial DNA copy likely underlie the pathogenesis of the disease state, including drug toxicity, cancer, and aging.
This video will demonstrate how to label newly synthesized mitochondrial DNA abbreviated M-T-D-N-A in dorsal root ganglion neurons that have been grown on sterile 12 millimeter glass cover slips. In a 24 well culture plate dilute the 10 millimolar stock of five ethanol two doxy uridine, abbreviated EDU from the Clicka EDO Microplate assay kit one to 100 in culture medium for a 10 XEDU stock. The 10 XEDU stock is then added directly into the medium in each well incubate the treated neurons for two to 24 hours at 37 degrees Celsius and 5%carbon dioxide in a fume hood.
Fix the neurons in 2%Paraform aldehyde for 10 to 15 minutes at room temperature. Wash twice in one XPBS for two to five minutes each wash. The fixed neurons can be stored in fresh one XPBS at four degrees Celsius for up to one month.
Prepare a humid chamber as described in the accompanying text. Use fine tipped forceps to transfer 28 fixed cover slips, including positive and negative EDU controls to sheets of hydrophobic paraform M inside the humid chamber. Reapply 200 to 300 microliters of one XPBS to cover the surface of each cover slip.
Prepare the click it assay kit as described in the text and the manufacturers and instructions to label mitochondrial DNA at 75 to 80 microliters of a permeable solution containing 0.1%tritton X in one XPBS to each cover, slip, cover, and incubate it room temperature for 10 minutes. Use a bulb transfer pipette with a 200 microliter tip affix to the end. To gently remove the liquid without losing cells.
Use a bulb pipette to gently flood the cover. Slip with 200 to 300 microliters of one XPBS to thoroughly wash out the previous solution. Wash each cover slip twice.
Pipette 75 to 80 microliters of fresh 1%hydrogen peroxide in one XPBS onto each cover slip. Incubate covered for 30 minutes at room temperature to quench endogenous peroxidase activity. Rinse twice with one XPBS as before freshly prepare the click at reaction cocktail as detailed in the accompanying text.
Pipe it up and down to mix. Do not vortex postfix the neurons with 75 to 80 microliters of click at EDU fixative per cover slip incubate room temperature for five minutes. During the postfix incubation, dilute the reaction cocktail with an equal volume of clicka EDU fixative.
Add the reaction cocktail to each cover slip cover to protect from light and incubate it room temperature for 25 minutes. Gently remove the reaction cocktail wash twice with diluted one x blocking buffer under an epi fluorescent microscope. Check for orgon green staining in nuclei when the EDU staining is complete.
Begin the termite signal amplification or TSA add 1%TSA block solution to each cover slip and incubate room temperature for 30 minutes. Briefly spin the antione green horse radish peroxidase conjugated antibody stock Prepared two to 24 hours in advance of TSA dilute the primary antibody one to 300 in TSA blocking solution and invert or pipette to mix tune knot vortex. Add 75 microliters to each cover, slip and incubate at four degrees Celsius overnight.
The next day, rinse three times with one XPBS at room temperature. Incubate the cover slips in the final wash for an additional 30 to 60 minutes to ensure that unbound primary antibody is removed. Prepare the IDE reaction according to the text portion of this protocol immediately before use.
Incubate the cover slips with the IDE reaction for 15 minutes at room temperature. Wash three times with one XPBS incubating in the final wash for 30 to 60 minutes as before under the microscope. Check for MT DNA labeling mount successfully stained neurons on glass slides with an ANTIFA mounting medium containing DPI shown here are representative differential interference contrast images of embryonic and adult dorsal root ganglion neurons that are typically used for analysis.
These schematic diagrams represent the procedure for labeling EDU in MTD NA with a green fluorescent signal. Mitotically active F 11 neuroblastoma cells serve as a positive control and illustrate the labeling pattern of EDU that was incorporated in nuclear and mitochondrial DNA in these representative fluorescence images overlaid over bright field. The green punctate signals show the amplified EDU signal incorporated into newly synthesized M-D-D-N-A of both embryonic and adult dorsal root ganglion neurons.
The EDU labeling procedure allows for subsequent immunofluorescent staining of neuronal markers such as neurofilament in red. These schematic diagrams represent the procedure for labeling EDU and MTD NA with a red fluorescent signal. Mitotically active F 11 neuroblastoma cells serve as a positive control and illustrate the labeling pattern of EDU that was incorporated in nuclear and mitochondrial DNA.
This schematic diagram represents the procedure for labeling BRDU in newly synthesized M-T-D-N-A with either a green or red fluorescent signal. While attempting this procedure, it's important to remember to run controls using mitotically active cells in order to check for the successful EDU labeling and cell nuclei before proceeding to the amplification steps to visualize MT DNA labeling Following this procedure. Other methods like standard immunofluorescence can be performed in order to answer additional questions like how mitochondria DNA synthesis is regulated in specific subpopulations of neurons or neuronal compartments Because of its development, this technique will pave the way for researchers in the field of neurobiology to explore the regulation of mitochondrial number in culture models of both normal physiology as well as culture models that simulate impaired disease.
Neurologic states.
