Terminal Transferase-Mediated dUTP Nick End-Labeling—TUNEL Assay: An In Situ Method to Detect DNA Fragmentation in Apoptotic Cells

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. TUNEL Assay

1. Euthanize animals that are showing clinical signs of chytridiomycosis and an equal number of Bd-negative control animals.
2. Dissect skin (dorsal, ventral, and thigh) samples from each animal. Fix the skin samples for 2 h in 4% v/v phosphate buffered formaldehyde. The short and consistent fixing time allows the tissues to be fully fixed, but also allows for effective and accurate immunohistochemistry staining. Then transfer to 80% ethanol until embedding for sectioning.
3. Embed skin in paraffin wax for histological preparation following standard methods. Briefly, the protocol is as follows:
   1. Dehydrate tissues in a graded series of ethanol and clear the ethanol with xylene. Embed the tissue in paraffin wax and place all three skin samples for each individual in one paraffin block.
4. Section skin using a microtome following standard histological preparation. Section the tissue serially at 5 µm and then affix it to hydrophilic glass slides. Place four serial histosections per skin type per slide. Make three slides per individual with serial skin sections, remember that each slide has four sections of each tissue affixed.
5. Stain the slides in the following order:
   1. Stain the first slide with hematoxylin followed by eosin counterstaining (H&E). This slide is to visualize the location of Bd sporangia within the skin.
   2. Stain the second slide following a commercially available TUNEL assay for histological preparation and follow the manufacturer's instructions, which are described below.
      1. First, deparaffinize the tissue sections in a coplin jar by washing the slides with three changes of xylene for 5 min per wash and follow with two changes of 100% ethanol for 5 min each wash. Follow with one wash of 95% ethanol for 3 min and then 70% ethanol for 3 min. Finish with one wash of PBS for 5 min.
      2. Pretreat the tissue with freshly diluted protein-digesting enzyme (proteinase K at a concentration of 20 μg/mL diluted in PBS), and add directly to the slide. Allow to incubate for 15 min. Wash with two changes of PBS in a coplin jar for 2 min each.
      3. Tap off the excess liquid and quench in 3.0% hydrogen peroxide in PBS in a coplin jar for 2 min at room temp. Rinse twice with PBS, for five minutes each wash.
      4. Tap off the excess liquid, then apply 75 µL/5 cm² of the equilibrium buffer directly onto the tissue on the slide. Incubate for 10 s. Tap off the excess liquid around the section and apply 55 µL/5 cm² of working terminal deoxynucleotidyl transferase (TdT) enzyme. Incubate in a humidified chamber for 1 h at 37 °C.
      5. After the TdT incubation, put the slide in a coplin jar of working strength stop/wash buffer, agitate for 15 s, and incubate for 10 min at room temperature. Wash the slide with three changes of PBS for 1 min per wash. Tap off the excess liquid.
      6. Apply anti-digoxigenin conjugate (rhodamine) that has been warmed to room temperature to the tissue, 65 µL/5 cm². Incubate in a humidified chamber for 30 min at room temp and avoid exposure to light. Wash with four changes of PBS for 2 min per wash. Tap off the excess liquid.
      7. Finish by adding 15 µL of 0.5 – 1 µg/mL DAPI (4',6-diamidino-2-phenylindole) in slide mounting medium to the slide, which acts as a counter stain. Then cover with a cover slip and seal with nail polish or rubber cement. Allow slides to dry in the dark as the assay is light-sensitive.
   3. Stain the third slide using the TUNEL assay and DAPI counterstain but use as a positive and negative control for each sample.
      NOTE: Of the 4 histosections on the control slides, the first 2 are positive control replicates and the last 2 are negative control replicates.
      1. For the positive controls, instead of step 1.5.2.2, pre-treat the tissue with DN buffer (30mM trizma base, pH 7.2, 4mM MgCl₂, 0.1mM DDT) and let incubate at room temperature for 5 minutes. Then dissolve Dnase I in DN buffer for a final concentration of 0.1ug/mL, and apply it directly to the slide. Incubate for 15 minutes at room temperature.  Then, wash the slide with 5 wash of dH₂O for 3 minutes each wash. Blot off the excess liquid. Then resume the TUNEL assay as directed in section 1.5.2.3.
      2. For the negative controls, do not add the terminal deoxynucleotidyl transferase (TdT) enzyme to those samples (as described in section 1.5.2.4).

2. Observe TUNEL slides immediately upon completion of the assay.

1. Take photos at random intervals along each skin section at 200X using a fluorescent microscope, using filters for both the rhodamine stain, which reveals the apoptotic cells and appears red, and DAPI which reveals all nuclei and appears blue, so that an overlay of cells can be generated. Ensure at least 100 cells are photographed per skin section per sample. Store the slides in a dark microscope box at -20 °C.
2. Count cells (blue DAPI stained) per image. Ensure at least 100 cells are counted per skin section. To reach 100 cells, count all cells within the image. If less than 100 cells are present in one image, count another image until at least 100 cells are reached per skin section per animal.
3. Next, count the number of TUNEL-positive cells in the same images (red rhodamine stained). TUNEL-positive cells, which indicate apoptosis and not necrosis or background, are single cells with clear cell edges (membranes are intact with no lysis). Sometimes the cells shrink to form apoptotic bodies.
4. Locate the areas counted in the TUNEL assay and analyze the corresponding regions on the H&E stained histosections. Confirm that the H&E-stained sections correspond to sites of Bd infection, which ensures Bd-infected sites are used when counting apoptotic cells in the TUNEL assay.