In situ hybridization

Hybridization of a cloned fragment of viral DNA to viral RNA (or DNA) in the infected cell can be achieved. The process is similar in broad outline to that for carrying out immunofluo-rescent analysis of antigens in a cell. In this type of hybridization, called in situ hybridization, the cells of interest are gently fixed and dehydrated on a microscope slide. Denatured probe DNA labeled with 3H- or 35S-labeled nucleosides is incubated with the cells on the slide. Following this, the slide is coated with liquid photographic emulsion that will detect radioactivity bound to the RNA or DNA of interest. The use of 3H- and 35S-labeled probes is favored because their decays are relatively low energy and the particle emitted is easily captured by x-ray emulsion near the site of its decay. Alternatively, a nonradioactive reagent can be incorporated into the probe DNA and detected with a secondary color or fluorescent reagent (FISH). When the micrograph is developed and observed, areas of RNA or DNA hybridizing to the specific probe are visible.

An example of in situ hybridization is shown in Fig. 12.10. For this study, human neurons from an individual latently infected with HSV were taken at autopsy and sectioned. One set of sections was incubated with radioactive viral DNA probe from a region of the genome not expressed during latent infection, and another was incubated with a probe covering a region of the viral genome that is transcribed into latency-associated transcripts during latent infection. The nature of these latent-phase transcripts is described in more detail in Chapter 17, Part IV. But here, it is necessary to point out that the positive hybridization signal is only seen with probes complementary to it.

Like immunohistochemical methods, in situ hybridization analysis can also be applied to larger scales. A histological section of a tissue or organ can be made, fixed, and then hybridized with an appropriate probe in order to locate areas where a specific viral transcript or viral genomes are being replicated. Indeed, the method can be applied to whole animals if they are small enough to allow sectioning. LP Villarreal and colleagues determined the effect of site of infection on the involvement of organs in which mouse polyomavirus will replicate in a suckling mouse using this method. An example is shown in Fig. 12.11. For this study, suckling mice were infected with polyomavirus by nasal or by intraperitoneal injection. After 6 days of replication, the mice were killed and carefully sectioned after freezing using a microtome, which is essentially a very sharp knife designed to cut thin sections of frozen or paraffin-embedded tissue. The slices were then placed on a membrane filter, stained, and hybridized with a radioactive polyoma-specific probe. The radioactivity was measured using a technique called fluorography, which is just a way of visualizing low-energy radiation.

Neuronal cell hybridized with clone 1 DNA

Neuronal cell hybridized with clone 3 DNA

Neuronal cell hybridized with clone 1 DNA

Neuronal cell hybridized with clone 3 DNA

  1. 12.10 In situ hybridization of human neurons latently infected with HSV. The trigeminal nerve ganglion was taken at autopsy from a middle-aged man killed in an automobile accident. The tissue was sectioned and individual slices incubated with labeled probe DNA from either region 1 or region 3 of the HSV genome shown in Fig. 12.9 under hybridization conditions. The left panel shows no hybridization; the dark spot in the neuronal nucleus is the nucleolus, which is the site of ribosomal RNA synthesis. The right panel shows positive hybridization due to the expression and nuclear localization of the HSV latency-associated transcript.
  2. 12.10 In situ hybridization of human neurons latently infected with HSV. The trigeminal nerve ganglion was taken at autopsy from a middle-aged man killed in an automobile accident. The tissue was sectioned and individual slices incubated with labeled probe DNA from either region 1 or region 3 of the HSV genome shown in Fig. 12.9 under hybridization conditions. The left panel shows no hybridization; the dark spot in the neuronal nucleus is the nucleolus, which is the site of ribosomal RNA synthesis. The right panel shows positive hybridization due to the expression and nuclear localization of the HSV latency-associated transcript.

Intraperitoneal infection with polyomavirus

Fig. 12.11 In situ hybridization of sections of suckling mice infected with polyomavirus. A stained section showing the location of major organs of the mouse is shown in the center. Fluororadiographs of sections showing tissues in which virus is replicating are shown above and below this section. (Photographs courtesy of L. P. Villarreal.)

Intraperitoneal infection with polyomavirus

Nasal infection with polyomavirus

It is very clear from the figure that virus inoculated in the nose replicates mainly in the lung, kidney, and thymus. By contrast, virus infected into the animal's peritoneum replicates efficiently in the kidney, brain, and bone marrow.

Was this article helpful?

0 0
How To Bolster Your Immune System

How To Bolster Your Immune System

All Natural Immune Boosters Proven To Fight Infection, Disease And More. Discover A Natural, Safe Effective Way To Boost Your Immune System Using Ingredients From Your Kitchen Cupboard. The only common sense, no holds barred guide to hit the market today no gimmicks, no pills, just old fashioned common sense remedies to cure colds, influenza, viral infections and more.

Get My Free Audio Book


Post a comment