1. The mice are 18-20 g upon arrival and are housed for a minimum of 1 wk prior to inoculation. The vast majority of our studies have been performed on male Swiss Webster mice. Balb/c mice behave similarly in our model. C57 black mice are significantly more resistant. Differences in susceptibility to the virus should be considered before selecting a mouse strain.

2. Input titer: selecting the input titer will depend on several issues, including the virulence of the viral strain(s) and the susceptibility of the mouse strains utilized. Our standard input titer with strain 17syn+ is 1-2 x 105PFU. This results in an infection rate of 100%, with a 10-20% rate of mortality in male Swiss Webster mice Utilizing this inoculum strain and titer, approx 25% of the neurons in the TG become latently infected, with a mean genome copy number of around 50. Over a period of 6 yr and hundreds of groups of mice, we have found that these results are extremely reproducible. However, when analyzing mutants with mutations in genes that reduce the ability of the virus to replicate in vivo, additional issues arise. First, replication at the body surface and in the TG is directly related to the number of latent infections established in the ganglia (13-16). Since the number of latent infections is directly correlated to reactivation frequency, any study with the goal of assessing the role of a particular gene or mutation in reactivation must be per formed on groups of mice with equivalent numbers of latent infections. By adjusting input titers of the mutant and rescue, the number of latent infections can be equalized, but this can only be done using methods to quantify latency accurately, for example (17).

3. It is critically important for in vivo studies to monitor the progression of the infection and determine the variability of viral replication from animal to animal. Viral replication at the body surface and in the ganglia is correlated with the establishment of latency. In turn, the size of the latent pool is correlated with reactivation efficiency. It is imperative that the investigator know the animal-to-animal variation with respect to those parameters critical to the facets of latency and reactivation being examined. The importance of minimizing animal-to-animal variation during primary infection cannot be overemphasized. This can be done with practice, even in outbred mice. We have found that independent analysis of three animals at each time point for viral replication at the surface site of inoculation and the TG on d 2, 4, 6, and 8 provides an excellent indicator of the quality and consistency of inoculation.

4. Temperature uniformity in the water bath is important. A forced/suction circulating bath is not ideal because the shed hair and fecal matter from the animals clog the apparatus. A bath with an internal stirrer rather than a pump eliminates this problem.

5. "Reactivation" is defined operatively and by definition requires the detection of infectious virus in a tissue in which infectious virus was previously undetectable. We define the term "initiation" of reactivation as detection of lytic viral proteins in a tissue in which lytic proteins were previously undetectable.

6. Mice are considered to be latently infected at times beyond 30 d post inoculation. In light of recent data (11,18), it may be prudent to delay for 40-50 d.

7. Those experienced in handling mice will have no difficulty with this. However, for those less experienced, this technique may help. Pick up the mouse as if you were going to give an intraperitoneal injection. Rotate wrist so that the mouse(abdomen down) is parallel with the benchtop. Take restrainer in free hand and put the open end of the restrainer up to the head of the mouse. Gently push the mouse forward into the restrainer while freeing your grasp. Adjust tail through slot of cap and secure cap.

8. As with all immunohistochemical procedures, optimum fixation conditions for any particular antigen/antibody combination must be determined empirically. In the case of this method, the low percentage of paraformaldehyde is important for maintaining the crosslinking of the tissue at a level consistent with penetration of primary and secondary antibody reagents.

9. Staining works equally well if the animal is fixed by perfusion rather than drop fixation of the tissue after dissection. Depending on the experimental design, one or the other method may be preferable. For example, we routinely perfusion-fix; however, if one TG is analyzed for infectious virus and the second TG is analyzed for viral protein, drop fixation is required.

10. Depending on experimental design, ganglia can be pooled and the procedure performed on all the ganglia from a given group in a single tube. Alternatively, ganglia can be placed in the wells of a 48-well plate and analyzed individually.

Fig. 5. Detection of HSV lytic viral protein expression in whole tissues. (A) Eyes d 2 post inoculation (pi) on corneas with strain 17 syn+. Arrow indicate regions of lytic protein expression on corneal surfaces. (B) Trigeminal ganglia 2 d pi. Neurons expressing lytic viral proteins are revealed (arrows). Increased protein expression in the TG on d 3 (C) and d 4 (D) parallels infectious virus titers.

Fig. 5. Detection of HSV lytic viral protein expression in whole tissues. (A) Eyes d 2 post inoculation (pi) on corneas with strain 17 syn+. Arrow indicate regions of lytic protein expression on corneal surfaces. (B) Trigeminal ganglia 2 d pi. Neurons expressing lytic viral proteins are revealed (arrows). Increased protein expression in the TG on d 3 (C) and d 4 (D) parallels infectious virus titers.

11. It is our experience with several antigen/antibody combinations that staining is enhanced by keeping the tissue at -70°C for several days. Again, this will have to be determined for the antigen/antibody utilized.

12. Incubating the primary antibody at 37°C can increase staining intensity.

13. Five hours is the minimum wash time. Increasing washing time and/or the salt concentration in the PBS from 0.15 M to 0.5-0.75 M can help reduce nonspecific binding.

14. The choice of detection strategies is limited by the ability of the enzyme conjugates to penetrate the tissue. HRP works well. Alkaline phosphatase-labeled secondary antibody reagents and biotin/avidin systems are larger in size and do not penetrate well.

15. Interpreting the results requires training. The appropriate controls must be analyzed along with test samples. A typical experiment should include TG from uninfected mice, TG from latently infected mice before treatment, and TG from the test group, i.e., from mice post hyperthermic stress. Do a trial run of the procedure on acutely infected ganglia (Fig. 5). Infected surface tissues can be included. Eyes shown in Fig. 5 were processed identically to the TG. Carefully compare uninfected and infected tissues under the microscope so that you will develop a sense of relevant vs irrelevant staining. The most troublesome "artifact" results from the inclusion of even very tiny pieces of lacrimal gland with the dissected ganglia. Ganglia should be checked thoroughly before proceeding with the assay to make sure that dissected ganglia are free of any contamination by this gland. As shown in Fig. 4, the lacrimal gland contains very dark brown to black structures, which, if freed during the staining process, can be interpreted as positive neurons. Neurons initiating reactivation will be extremely rare (see Subheading 1. for references).

16. We have utilized this method successfully with a number of different antibodies, including antibodies recognizing specific lytic viral proteins as well as host cell proteins.

17. Pressing ganglia between slides allows viewing at the higher magnification required for identifying and counting positive neurons. Other tissues, such as eyes and snouts or TGs during lytic infection, can be viewed under a dissecting microscope without pressing (Fig. 5).


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