New technique for improving staining in older FFPE sections.

The small biotech where I am employed frequently works with with FFPE specimens that are at least 10 or more years old, and 20+ year old specimens are not particularly unusual. Any histologist can tell you that the performance of older FFPE blocks and sections used in IHC and nucleic acid hybridization studies gradually degrades with time. The logical assumption is that the target molecules are decaying and losing their affinity for the probes sent to detect them. Various antigen retrieval techniques and signal-boosting protocols have been used to try to get older tissues to take up stain more efficiently, but their usefulness seems limited. We have taken a different approach and have been experimenting with ways to "restore" older FFPE tissue samples so that the specimen stains just as it did when it was first fixed. The process we are developing seems to improve staining in both IHC and ISH studies. It also seems to help optimize IHC studies such that a uniform staining protocol can be used with similar sections of any age. I can't say too much about the details of the proprietary process that we are still developing, but I can show you some before and after images to give you an idea of how effective it is.

The first pair of images below simply illustrates the problem. The image on the left was taken in 1996 and shows HPV in situ analysis. The image on the right is the same tissue analyzed with the same probes in 2012. Nothing controversial here, just a the typical loss of signal strength that most scientists would predict in 16 year old tissue.

(Click on the image to enlarge)

In the following pairs of images we see serial sections taken from tissue blocks of various ages that have been used for  either IHC or ISH studies. In each pair, the image on the left shows the staining one might typically expect from an old section or a section from an old block. The image on the right is a similar section that was pretreated with our proprietary process, then subjected to the exact same analysis as the section shown on the left.

(Click on the images to enlarge)

These results are quite striking. So striking, in fact, that we are not sure that we totally believe them ourselves. Although we have had the effectiveness of this process confirmed by at least one third-party laboratory, we are currently actively seeking additional organizations with experience in IHC to help us validate this procedure.

If you are a lab that is struggling with poor staining in older slides, please contact me to see if we can enter into some kind of mutually beneficial collaboration. Ideally, we would set up a carefully controlled blind study where you send us pairs of problematic slides, we would treat one of the pair and send them back to you. We won't tell you which one we treated. You then stain and examine them as normal, and let us know if you see a distinct difference between them and also with controls kept at your location. Naturally there would be no charge for this. Ultimately we may be interested in commercializing this process, but first we need to be absolutely sure that it really works the way we think it does.

Separating out different color wavelengths to better understand target distribution

The nuance multispectral imaging system from PerkinElmer is pretty cool. Basically it's just a camera with some spectral filters that sits on top of your microscope, and some smart software that can make sense of the captured image. The system lets you isolate the color signal of specific stains, filter out the noise that comes from autofluorescence, figure out which cells have taken up which stain, and impartially calculate the strength of the staining response. Best of all it can use some wavelength addition and subtraction calculations to highlight areas that have taken up more than one stain. 

In a recent study conducted in our CLIA certified lab, we used this system to show co-localization of a particular protein and the mRNA transcript from which that protein was translated. The 4 images below are of the same region of a human prostate section. IHC was used to stain a smooth muscle cytoskeletal protein called "smoothelin". ISH was then used to stain the same material so that we can see the mRNA transcripts from which the protein was translated. 

The first image shows the presence of antibody stained protein as brown, the blue stain is the mRNA. Note the localization of the mRNA signal to the stroma (long arrow) and the lack of signal in the epithelia (short arrow). In the first image (brightfield microscopy), it's sort of hard to see if the brown and the blue stains are really tightly co-localized.

In the next pair of images, fluorescence microscopy is used to separately show the smoothelin protein (green) and the mRNA transcript (blue).

Finally, in the last image, the nuance system helps us to see BOTH types of staining simultaneously, with the dual stained regions shown as yellow. This makes the co-expression much easier to see. For more information on the Nuance system here.

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?