The overall goal of this procedure is to allow for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh frozen tissue sections. This is accomplished by first collecting cryostat sections from the organ of interest. The second step of the procedure is to perform the Cox Histo Chemistry, which is followed by the SDH Histo chemistry.
Finally, the tissue sections are dehydrated, mounted and cover slipped. Ultimately, results can show the amount of mitochondrial dysfunction through the amount of cellular blue staining. Hi.Today we're gonna talk about the Cox SCH double labeling HISTOCHEMICAL technique.
And while this method can be applied to study mitochondria diseases, it can also provide insight into aging and age related disorders. After removing the brain from an animal that has been sacrificed in accordance with ethical permit, rapidly freeze it on dry ice. Frozen tissue can be kept at minus 80 degrees Celsius, wrapped in aluminum foil until ready to section.
Prepare the tissue for cryosectioning by placing the brain on a cryostat chuck and surrounding it with embedding compound. Quickly place the chuck into the cryostat and collect 14 micron sections at minus 21 degrees Celsius. Thaw the sections onto slides using a heating block, and then allow the slides to dry at room temperature for one hour.
Place the slides in a slide staining chamber with wet paper strips. Because reaction times are important in this assay, it is recommended to process a maximum of 10 slides per experiment under a chemical hood. Prepare incubation, medium containing dab and cytochrome C in PBS and vortex.
Quickly add two micrograms of bovine CDEs to the medium and mix well by vortexing. To break up all the grains of CDEs still working under the hood, apply 150 to 200 microliters of incubation medium to each slide using the pipette tip to spread evenly onto all sections. Incubate the slides for 40 minutes at 37 degrees Celsius.
After 40 minutes, remove the incubation medium from the slides. Wash the slides four times in PBS for 10 minutes each time. After washing the slides, return them to the slide staining chamber with wet paper strips working under a chemical hood.
Prepare the NBT solution in PBS taking care to shield the PMS from light vortex the solution quickly apply 150 to 200 microliters of the NBT solution to each slide using the pipette tip to spread evenly onto all sections. Incubate the slides for 40 minutes at 37 degrees Celsius after 40 minutes, remove excess solution from the slides. Wash the slides four times in PBS for 10 minutes each time.
Dehydrate the slides for two minutes each in the following sequential concentrations of ethanol. 70%70%95%95%and 99.5%Finally, allow 10 minutes in an additional 99.5%step. Place slides in xylene for 10 minutes mount with LAN and apply cover slips.
Allow the slides to dry overnight or at least one to two hours in a ventilated area here. Brain sections from wild type and prematurely aging. MT.DNA Mutator mice were sequentially labeled for COX and SDH activities.
Normal Cox activity indicated by the dark brown staining is seen in the hippocampus of wild type mice. Cox deficiencies indicated by the blue staining were revealed in the hippocampus from MT DNA mutator mice at 12 and 46 weeks. There was a further decrease in Cox activity by 46 weeks of age in M-T-D-N-A mutator mice suggesting widespread exacerbation of respiratory chain dysfunction.
Inadequate incubation times of 10 and 25 minutes resulted in reduced deposition of the brown dab reaction product. The shortened incubation times allowed for the formation of the blue Foran end product during the SDH incubation misleadingly suggesting the presence of cells with COX deficiencies cover slipping. The slides during the COX incubation also resulted in inaccurate formation and deposition of the DAB reaction product.
Although Cox and SDH activities can be individually labeled as shown here, the sequential labeling has proved to be advantageous in locating cells with mitochondrial dysfunction. An example of a specificity control for COX and SDH activities in brain from a wild type mouse shows negative labeling. So don't forget that working with the chemicals in the Cox SCH double labeling is the chemical protocol are extremely hazardous and precautions such as wearing proper lab attire and a mask working under the chemical hood, and also disposing of the incubation medium should be done every time you perform this assay.
