This video shows how to perform metabolic mapping. A procedure to quantitate the activity of specific enzymes called dehydrogenases. The cells adhere to a microscopy slide or a frozen tissue.
Decades ago we developed a whole series of metabolic mapping techniques then called enzymistochemistry to detect activity in tissues and cells under the microscope. However metabolism became obsolete during the genetic revolution at the end of last century. But now, metabolism is hot again.
Look, for example, at the cancer cells. The metabolism is now considered to be a therapeutic target, and therefore, activity determinations in cells and tissues is hot again. A key component of the reaction buffer is polyvinyl alcohol, or PVA.
This is dissolved in phosphate buffer at a concentration of 18%PVA is dusty and toxic when inhaled, so be sure to weigh PVA under a fume hood. Because of the high PVA concentration in the phosphate buffer, heat the reaction buffer bottle au bain-marie while stirring. Be sure to screw the cap loosely on the bottle or the bottle may explode.
It may take up to 15 minutes before the PVA is completely dissolved in the phosphate buffer. If so, the solution should be transparent with small air bubbles that were caused by the stirring. The viscous PVA solution can be pipetted into a reaction buffer tubes using a wide pipette.
Keep the tubes at at least 40 degrees Centigrade or higher to prevent the PVA from solidifying. One milliliter of PVA solution is good for about four cytospins, or two tissue preparations. For the other components of the reaction buffer, the yellow nitroblue tetrazolium chloride also called nitroBT, is dissolved in a mixture of ethanol and dimethylformamide, while heating it in a glass vial in the Bunsen burner.
The solution should not come to a boil or the volume will decrease too much. So do not leave the vial too long in the flame and shake regularly. Keep it on ice after being dissolved and proceed to the next step quickly as the dissolved nitroBT perishes rapidly.
NitroBT is a yellow powder that will lie on the bottom of the glass vial containing ethanol and DMF when it is not kept dissolved. You can check every few times if the nitroBT is dissolved. The solution will then be yellow transparent.
When dissolved, the nitroBT can be pipetted in the vials containing the PVA solution. Stir gently using a thin spoon or a thin spatula to mix the nitroBT and the buffer. Do this for all components of the reaction buffer step by step that are listed in table one of the full text.
Try not to make too much air bubbles while stirring the reaction components into the PVA solution. The trick is to stir continuously while not taking the spoon or spatula out of the solution. The electron carrier, methoxy phenazinium methyl sulfate, is to be protected from light and dissolved into the reaction buffer last.
The red color of the MPMS is convenient to double check whether all components are dissolved correctly. While preparing the reaction buffer and stirring the components into it, prewarm the slides for 50 minutes in an incubator containing warm water. Keep a low water level inside the incubation chamber.
With the lid closed, this will keep the reaction environment humid. With the lid open, the water will keep the slides at a warm temperature. Apply the reaction buffer to the cell or tissue preparations using a glass pipette.
Do not worry about small air bubbles because these will flow to the surrface and it will not interfere with the reaction in the cells or tissues. Keep the lid of the incubation chamber closed during the incubation time. This will make sure the reaction occurs in a humid environment to prevent the PVA buffer from drying up.
Most dehydrogenase reactions in metabolic mapping experiments occur at 37 degrees Centigrade. Incubate the slides in the reaction buffer for a prespecified amount of time, which can be found in table one of the full text article. One can readily see whether or not the reaction worked as the cells, or tissue, will turn dark blue or purple, due to the dehydrogenases'activity.
Which ultimately turns to colorless insoluble nitroBT to the dark blue formazol, which precipitates at the site of dehydrogenase activity. The enzymatic reaction is stopped in three steps. First, the excess incubation medium is to be tapped off on a tissue.
Second, the incubation medium is to be washed off of the microscopy slides using mechanical washing in a phosphate buffer of PH of 5.3 at 60 degrees centigrade. Third, the microscopy slides are to be left in fresh phosphate buffer of PH 5.3 at 60 degrees Centigrade for 20 minutes. Fourth, the phosphate buffer can be rinsed off using tap and then distilled water.
One can readily see the reaction buffer coming off. The stopping buffer's low PH helps in stopping the dehydrogenase reaction inside the cells. Place the slides in fresh stopping buffer once or twice depending on how much slides you are processing at a time.
Also rinse the slides with tap water and finish with distilled water. Now, the slides can be dried on a slide warmer and enclosed. Carefully dry the back side of the slide by wiping it and place on a slide warmer preheated to 50 to 60 degrees Centigrade.
When the slides are dry, a glycerine glycerol solution and cover slips can be used to enclose and preserve slides. Save the slides in the refrigerator until proceeding with image acquisition. This is a color photo micrograph of a representative tissue section on a glass slide that was processed in the experiment shown in this video.
This is a glioblastoma section and lepto dehydrogenase is the enzyme that was investigated. Every dehydrogenase produces entity PH and every entity PH molecule converts one molecule of nitroBT, the yellow tetrazolium salt, to one molecule of formazol, the purple precipitate shown in this image. One can see high lactate dehydrogenase activity intracellularly in the tumor cells shown as dark purple.
The blood vessels have lower activity, and are white. This is a monochromatic image of the same region of the slide. Which can be used to quantify the enzyme activity as described in the protocol.
The more formazan is produced, the darker the pixel, and the higher the absorbance. The absorbance can then be converted to picomoles of entity ph that were generated. So metabolic mapping is easy to do.
It's very fast and it's cheap. So use it in your research.
