Detection of Reactivation in the TG see Note

Peak infectious virus production occurs in the TG at 22-24 h after hyperthermic stress (5).

3.3.1. Harvesting Tissues

Animals are euthanized according to the investigator's protocol. Sterile instruments and technique should be employed when harvesting tissues for detection of infectious virus. If surface tissues will be analyzed, these should be removed prior to wetting the animal with ethanol. Eyes are easily removed with forceps and whisker pads with small curved scissors. Wet down mouse fur with ethanol. To remove the TG, open the skull and peel back the brain (Fig. 3). The TG lie to the right and left of the pituitary (Fig. 3B).

For detection of infectious virus, ganglia are placed upon removal in ice-cold sterile 1.5-mL tubes containing 1 mL of media and maintained on ice.

3.3.2. Homogenization

1. Place on ice sterile 2.0-mL straight-wall ground-glass tissue homogenizers (Radnoti) and chill.

2. Transfer the media and the tissue to the base of the grinder, and fit the pestle into a flexible chuck adapter installed into a stirring motor (IKA-RW15).

3. Homogenize the tissue with 7-10 strokes set at 2.5 (5 g) on ice.

4. Rinse the homogenate remaining on the pestle with a small amount of media into the grinder, and use a sterile plugged 9-inch Pasteur pipet to transfer the homogenate from the grinder to a 1.5-mL tube.

5. Centrifuge homogenates (2,250 g at 4°C) for 5 min, place on ice, and immediately plate onto indicator monolayers.

3.3.3. Plating Homogenates

1. Prepare indicator cell monolayers the day before. Monolayers in 6-well or 60-mm plates should be just confluent at the time of use.

2. Pipet the entire homogenate onto the monolayer and absorb with rocking for 2 h (37°C, 5% CO2).

3. Remove the homogenate, rinse the plates twice with media, overlay with media containing 1% methylcellulose, and incubate at 37°C in 5% CO2 until plaques are a convenient size to count under the dissecting microscope (48-72 h).

4. Remove the overlay from plates, and rinse them three times with PBS before adding crystal violet.

3.4. Detection of the Initiation of Reactivation (Detecting Expression of Lytic Genes in Latently Infected Ganglia)

1. Day 1. Euthanize animals according to investigator's protocol. For fixation, proceed as follows:

a. Dissect TG as described above.

b. Immediately upon removal, place tissue in 0.5% paraformaldehyde in PBS and incubate at room temperature with gentle agitation (nutate; Adams Nutator) for 2 h. Tubes of 1.5 mL work well (see Notes 8-10).

c. Remove paraformaldehyde. (Follow your institutional biohazard guidelines for disposal.)

d. Rinse in PBS three times for 15 min each.

e. Remove final PBS wash, and add methanol containing 20% DMSO.

f. Nutate overnight at room temperature.

2. Day 2: Endogenous peroxidase pretreatment I.

a. Remove methanol/DMSO, and nutate tissue for 1 h in methanol containing 20% DMSO and 10% H2O2.

b. Remove methanol/DMSO/H2O2 solution, and rinse tissue in 100% methanol 3 X 15 min. on nutator.

c. Store tissue in 100% methanol at -70°C overnight or longer (see Note 11).

Fig. 4. (continued)

3. Day 3: Endogenous peroxidase pretreatment II (see Note 8).

a. Bring tissue in methanol to room temperature. Remove methanol and rehydrate by incubating in PBS 3 X 15 min each on nutator b. Incubate tissue for 2 h at 37°C in PBS containing 0.002 MNa azide, 0.02 M glucose, and 100 |g/mL GO (12).

c. Remove GO solution, and rinse in PBS 3 X 15 min, on nutator.

d. Remove PBS and add primary antibody, rabbit anti-HSV (Accurate) diluted 1:3000 in PBS containing 2% BSA, 5% NHS, 5% DMSO, and nutate at room temperature or 37°C overnight (see Note 12).

4. Day 4: remove primary antibody.

a. Rinse in PBS 5 X 60 min each (total 5 h) on nutator (see Note 13).

b. Remove PBS and add HRP-labeled antirabbit antibody (Vector) diluted 1:500 in PBS containing 2% BSA, 5% NHS, 5% DMSO, and nutate at room temperature overnight (see Note 14).

5. Day 5: remove HRP-labeled antibody.

b. Remove last PBS wash, and rinse once in TBS.

c. Incubate in 0.1 M Tris-HCl, pH 8.2, containing 250 |g/mL DAB and 0.004% H2O2 solution. Typical development time is 5 min; however, this will need to be determined empirically.

d. Remove DAB solution and rinse twice in ddH2O for 15 min each.

e. Remove ddH2O, and nutate in 100% glycerol overnight.

f. Press ganglia between two glass slides, tape, and examine under a microscope (Fig. 4); see Notes 15-17).

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