The overall goal of this procedure is to selectively label active phagocytic cells, which clear out cell corpses in stroke. This is accomplished by first preparing components for the Tite based signal amplification reaction. The second step is to prepare the sections.
Next, the labeling reaction is performed using the enzyme vaccinia, topoisomerase and liable fluorescent probes. The final step is IDE based fluorescent signal enhancement. Ultimately, fluorescence microscopy is used to assess phagocytic clearance of dead cells in tissue sections of focal brain ischemia.
The clearance of dead cells by phagocytes plays an important role in brain recovery after ischemia. The presented method determines the efficiency of this process by labeling only those pego sizes which actively remove dead cells. Though this method provides insight into the ischemic brain, it can also be used to study ischemic injury in other organs and tissues.
Where FA acidic clearance of dead cells is important. Although the IDE signal amplification or TSA step occurs in the final stage of labeling, preparing for this reaction can take more than an hour. It is therefore more convenient to make the TSA reagents First.
The first step is to make the fluoro four TYROM stock solution at 300 microliters of DMSO to the tyrom reagent files included in the TSA labeling kit. Next, prepare the TNT wash buffer to completely dissolve the blocking reagent. When making the TNB blocking buffer slowly add it in small increments while stirring heat the solution gradually to 55 degrees Celsius.
With continuous stirring, the solution will appear milky and should be brought to room temperature before using aliquot and store at negative 20 degrees Celsius for long-term use for this procedure, slides are prepared ahead of time by mounting five micrometer thick sections. Cut from formalin fixed tissue. First, treat the sections with xylene for 15 minutes, rehydrate the sections through a series of ethanol washes, followed by two washes in water.
Next, make the proteinase K working solution by diluting a stock solution to 50 micrograms per milliliter in PBS. The most difficult part of this experiment is determining the ideal length of time for proteinase K digestion controls with both shorter and longer digestion times might be needed to ensure success Depending on the size and type of tissue. Add the appropriate volume of protein ACE K working solution to the sections and incubate for 15 minutes at 23 degrees Celsius.
After the incubation, wash the sections in distilled water two times for five minutes each. While the sections are soaking, prepare the labeling mix containing the active probe. To prepare the active probe solution, combine the oligonucleotides with water one molar triss, HCL and the vaccinia topoisomerase supplied in the kit mixed gently by pipetting and incubate at room temperature for 15 minutes to allow for probe activation.
After the second wash period, take the sections from the jar and aspirate off the remaining water. When ready, apply the active probe solution prepared earlier cover. Slip the sections and incubate them for 15 minutes at 23 degrees Celsius.
After the incubation, dip the slides in water to gently remove the cover slips. Wash the slides in water three times for five minutes each. After the last wash, cover the tissue sections with TNB blocking buffer and incubate the slides in a humidified chamber for 30 minutes at room temperature.
During this time, prepare a one to 100 dilution of stripped and horse radish peroxidase conjugates. S-A-H-R-P in TNB blocking buffer, retrieve the slides and aspirate the TNB blocking buffer away. Before applying the S-A-H-R-P solution, use enough reagent to cover the entire tissue section.
Once covered, incubate the slides for 30 minutes at room temperature After the incubation, wash the slides three times for five minutes each. In TNT wash buffer at room temperature, prepare a one to 50 dilution of fluoro four TY IDE stock in one x amplification diluent immediately before use. Pipette the solution onto the slides completely covering the tissue section.
Since the solution cannot be reused, discard any unused portion remaining. Incubate the slides at room temperature for three to 10 minutes. Monitor the fluorescence.
Increase under the microscope until the first hints of structures are visible at this time. Quickly proceed to the wash step. Wash the slides three times for five minutes each.
In TNT Wash buffer at room temperature with agitation. After washing, add the anti fading solution with DPI and cover slip. Observe the results under a fluorescence microscope.
These images show two examples of PHA acidic clearance. The nuclear stain dappy is shown in blue fluorescence and the liable vac topo probe is shown in green fluorescence diagrams of the events presented in these images show nuclei of phagocytes and tic engulfed extra nuclei in various stages of digestion. Once mastered, this technique can be done in about three hours if it is performed properly.
After watching this video, you will know how to use liable fluorescent probes in tissue sections. You will also be able to selectively label active ides clearing out dead cells in ischemic brain.
