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The Full Story

About the colors


Small samples deriving from different organs (skin, nerves, spinal cord, brain, muscle, adipose tissue) are collected, prepared and conserved  for histology following specific procedures (standard protocols)  to optimize the following “colouring” method.

This technique is known as immunohistochemistry because is based on the link between antibody and antigen (“immuno”) in histology (“histo”) of the molecules (“chemistry”).

Perhaps it is not a coincidence that the root of the word histo-logy derives from the Greek  ἱστός (histós), canvas…

But, we proceed by degrees and I explain everything  or at least I try!


Tissue is my blank canvas.

Canvas preparation: After tissue conservation, the sample must meet of requirements. It must be sufficiently thin (depending from the kind of analysis, from 10 to 40 micron of thickness). An instrument called cryostat allows to obtain very accurate slices. Slices can be mounted on a lab glass (slide) or let free-floating in a well with physiological solution.

Primary antibodies are my brushes.

Choice of the brushes: Depending on our interest, proteins (molecules) to mark (to paint) are identified  and specific antibodies (primary antibodies) will be utilized.


The primary antibodies specifically recognized the protein of interest (that serves as antigen) and dock it.  The concept is the same of the immune system: when our immune system recognizes an antigen of a pathogen (i.e. a bacterial protein), it release antibodies against this, which bind the intruder.

 A solution containing different (up to 3) primary antibodies is prepared and samples (on glasses or in well) are exposed (immersed) and left to incubate (to act) overnight or more.

IMPENETRABLE impenetrable barrier glial scar.jpg

Secondary antibodies are my palette.

Choice of the nuances: Primary antibodies are not “colored”, they serve as “tag” to indicate at the secondary antibodies where to bind.

On the same immune principle of the primary antibodies, the secondaries bind the antigen, that this time is not the protein of our interest but the primary antibody which, in this occasion, serves as a tag.

We will obtain a sort of sandwich: tissue protein is linked by primary antibody which in turn is linked by secondary antibody.

A peculiarity of the secondary antibodies is that they are fluorescent because they are joined to a fluorochrome.

Fluorochromes “are photoreactive chemicals that can absorb energy via the interaction of an orbital electron in the molecule's atomic structure with a photon of light”.


Basically, fluorochromes have spectral features and when stimulated by a specific wavelengths (by means of laser) they emit a light in a different wavelengths. For example, if we stimulate the fluorochrome fluorescein with a light at 488 nm (contained in the wavelengths of blue range, 435-500 nm) it will emit a fluorescence in order of 521 nm, in the range of green.

After an accurate evaluation, 3 different fluorochromes will be chosen and diluted in a solution, distributed on the assays which will be let to incubate for some hours. At the end, the samples are incubated with  DAPI (4′,6-diamidino-2-phenylindole) which is a blue-fluorescent DNA stain, utilized to mark the cells nuclei.

After this long procedure, I feel a bit like a wizard that following a magic formula, put in a pot secret ingredients without to have a direct feedback of the result.

Only passing hours and hours with an eye in a confocal microscopy, I can admire the vivacity and brilliance of every single color and their matching,  in determining an overall harmony even when what is shown is the representation of catastrophic degenerative disease. It’s like when you are fascinated, although scared, by the powerful of a blazing fire.

By confocal microscopy I can see across, but my eye and my feel make me see what and how.  

Few details on Fluorochromes 

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