Tuesday, October 16, 2012

In-situ hybridizations and FFPE slides.

Here's an example of the difference between what you could do with an FFPE, H&E slide 15 years ago versus today. This illustrates WHY archives of FFPE tissue have become so much more useful in recent years. In this example we are looking at a sagittal section of an embryonic mouse:

Abbreviations: Br – brain; DMO – dorsal region medulla oblongata; IC – internal capsule; K – kidney; Li – liver with blood vessels; Lu – lung; Ma – mandible; NCh – nasal chamber; NPhD – nasopharangeal duct; PhU – phallic part of urethra; (s) – sense. 

So, you can see that the mouse has cells, tissues, organs, the usual, yup, it's definitely a mouse, and honestly that's about all you can tell from the above image.

Now imagine you are interested in a specific gene, and you want to know where that gene is most actively expressed. Today you can do that using a technique called in situ hybridization (ISH).  The image below was taken from a study of a gene I will simply call "GENE X"  which is believed to code for a transcription factor protein. In humans, GENE X mutations cause severe diseases including neonatal diabetes mellitus.  The purpose of this ISH analysis was to localize GENE X mRNA at the anatomical and cellular levels in order to help scientists learn something of its likely function.  The formaldehyde fixed sections were mounted on glass slides then hybridized with 35S-labeled cRNA probes. Patterns of gene expression were analyzed by x-ray film autoradiography and  darkfield illumination. The image below is of the same section as the one above.

The results provided evidence for the presence of GENE A mRNA at various concentrations in several tissues including specific brain regions, structures related to the nasal chamber, lung, liver blood vessels, kidney, ureter and pancreas. In the pancreatic islets, the hybridization intensity was comparable to that of the kidney cortex. I won't bore you with all the details of what this experiment suggests, but the point here is that now the scientist has the option to ask questions about what specific genes might be doing in specific places and at specific times during the development of the organism. Pretty neat huh?

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