Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

This method can help answer key questions in the biology field such as activity of enzymes at different locations within tissues. The main advantage of this technique is that it allows the researcher to detect enzymatic activity directly in the tissue samples. To begin this procedure, remove the mouse's hair with an animal hair trimmer.

Make an incision on the skin of the back using scissors. Then, lift the skin up and cut it away. Next, fill the Cryomold with freezing reagent compound.

Using needle nose forceps, place the skin sections into the field's Cryomold. Orient the skin sections so that the cross section of the skin from the epidermis to the dermis, hypodermis and muscle is visible. If possible, mount the experimental and control mice together in the same Cryomold, so they can be sectioned on to the same slide and processed together.

Take care not to create bubbles. Place the Cryomold on a flat surface of a block of dry ice in an ice bucket. For storage, transfer the Cryomold into a negative 80 degree Celsius freezer.

Samples can be maintained in the freezer for approximately three months without significant loss of enzymatic activity. For sectioning, using a cryostat set at negative 20 degree Celsius, slice the tissue to create seven to 10 micrometer-thick sections for the best skin morphology. In this procedure, establish the set of slides to be tested, which includes a control slide on which NAD will be withheld, another control slide on which the substrate lactate will be withheld, and a positive control slide from a frozen tissue block previously investigated.

Briefly fix the slides containing the skin sections with 4%formalin for five minutes. Then, wash the slides with PBS. Next, vortex the reagents, including the NADP, PMS, nitrotetrazolium blue chloride, and lactate together for lactate dehydrogenase activity staining in an appropriately sized tube.

Next, prepare a second solution in which all reagents are present except NAD. Subsequently, prepare a third solution in which all reagents are present except lactate. To incubate the slides and staining solution or control solutions, dip them into the solution at the same time.

Then, incubate them in a humidified environment at 37 degree Celsius in the dark. Monitor the conversion of the staining solution from clear to blue. When the samples have reached the desired level of blueness, stop the reaction by removing the staining solution and rinsing them with PBS.

Afterward, a counterstain on to the slides when the slides have reached an appropriate level of blueness. Counterstains that turn the nuclear red or green will provide good contrast with the blue created by the Formosan precipitant. Then, mount the slides with aqueous or non-aqueous mounting medium.

One to three drops of mounting medium is sufficient depending on the size of the cover slip. Subsequently, image the slides under a light microscope. Take photographs of experimental and control samples, and all negative controls at 10X, 20X, and 40X magnification.

The intensity of staining will increase over time as the same is incubated with the staining solution. As shown here, high levels of lactate dehydrogenase activity were observed in the hair follicle stem cells at the base of the hair follicle. Control samples that did not receive the lactate substrate did not show staining.

These findings were corroborated by fluorescent activated cell sorting of skin for hair follicle stem cells and confirming high lactate dehydrogenase activity in the stem cell compartment with activity on sorted cells. Based on the findings that hair follicle stem cells have high lactate dehydrogenase activity, cultured embryonic stem cells were tested. Mouse embryonic fibroblast serving as a feeder layer were analyzed in the presence of embryonic stem cells or alone.

High lactate dehydrogenase activity was observed in the embryonic stem cells as compared with the mouse embryonic fibroblast as shown in different time points. The staining protocol was also applied to freshly isolated tissue. Mouse aortas were dissected.

Samples were incubated with Hank's balanced salt solution containing 0.5%Triton X for 10 minutes. Then, with the lactate dehydrogenase staining solution for 10 minutes. The images here show lactate dehydrogenase solution with and without lactate.

While attempting this procedure, it's important to remember to use frozen tissue slices with intact enzymatic activity and the frozen blocks should not be too old. If prepared sections are being stored, the sections should not be allowed to dry out. After watching this video, you should have a good understanding of how to visualize lactate dehydrogenase activity in the mouse skin.