The alchemy of impregnation

Silver is the most commonly used metal to impregnate tissue structures, but it is by no means the only one. Although relatively rare, we can also use gold to detect substances such as amyloid or to mark astrocytes with Cajal gold sublimate (gold and mercury chloride).

Of course, not everyone sees methods of depositing metals other than silver or gold as impregnation in and of themselves. It depends to some extent on what constitutes "impregnation" and how we define it. The term is interpreted fairly broadly and would encompass almost any method in which metal is selectively attached in some way to any tissue structure and then made visible in some way. This includes compounds such as osmium tetroxide for triglycerides, copper for fatty acids, and both colloidal iron methods for acidic mucopolysaccharides and the Schmorl ferrocyanide reduction method for melanin and enterochromaffin. All of these metal deposition techniques demonstrate it either physically or chemically.

Although metal ions are also used to create dyes, they are essentially either carriers of the dye or simply combine with it and change its color. The main focus is still on the dye, so they are seen as a variation of staining. In impregnation, it is the metal itself or one of its insoluble salts that forms the final visible component, i.e. all attention is focused on the metal.

Silver

Impregnation with silver ions is quite well known as a method and today I will not focus on it. While I'm pretty sure terms like "argentaffin" and "argyrophiles" can give some thought, I will refer to them later when it comes time to develop this methodology.

Silver impregnation - Diapath.com

Gold

Gold impregnations are no better understood than silver impregnations. They were developed empirically and remain perceived as such. Usually experience and knowledge of techniques are required.

Silver and gold impregnation are older staining techniques that were widely used years ago. Even today, gold impregnation is used occasionally to observe detailed biological structures and processes, such as intercellular connections and nerve cell processes. Depending on the method used and the material (silver / gold), the end result can be brown, gold or black.

Proliferation of reactive astrocytes. Cajal Gold sublimate. (1)

Osm

Lipid osmation is fairly well understood. Osmium tetroxide dissolves or chemically reacts with lipids, depending on the type of lipid. During dehydration, the dissolved osmium in the lipids is reduced to another osmium compound, possibly the lower oxide. When working with solvents such as xylene, lipids are dissolved, leaving a black deposit of osmium where they used to be. These lipids, which react chemically with osmium tetroxide, are fixed and are not affected by any chemical reagents. They remain in the paraffin embedded tissue and are blackened by reactions with osmium tetroxide, which also fixes them. Both processes occur at the same time, so in both cases the presence of lipids is manifested by black osmium deposits.

Unfortunately, osmium tetroxide has two very serious drawbacks. It is quite toxic and very expensive. For these reasons it is mostly used in situations where frozen sections are impossible to make in order to dye Sudan. It must be remembered that this is really the only fixative that allows the preservation of lipids during the paraffin infiltration process, although it is also important for the preservation of lipid membranes and structures such as mitochondria.

Tetroxide is expensive and toxic… but also capricious. It is a fixative that infiltrates tissue very slowly. To the extent that by fixing the top layers of the tissue section, it blocks the way for further penetration itself, therefore most often it is required to take very thin sections, and if possible even small ones, so that the entire tissue can be well persoaked. As a rule, good tissue infiltration with a thickness of 2 mm takes approximately 24 hours. It doesn't matter if we put the tissue in the incubator, because it's the heavy osmium tetroxide fault. That is why it is better to carry out the whole process in the refrigerator to maintain the better structure of the tissue itself.

How does osmium tetroxide work?

1. According to Professor Baker, it likely binds proteins through the tryptophan and histidine side chains, and possibly blocks the amino groups, as staining with acid dyes is later very difficult. It does not seem to have any effect on DNA. Carbohydrates are largely intact too.

2. After fixation in buffered formalin for at least 24 hours, rinse thin tissue sections overnight under running water to remove all traces of fixative.

3. Rinsed tissue should be rinsed with several changes of distilled water for an hour or more.

4. Place the tissue in 0.5% osmium tetroxide dissolved in distilled water for 24 hours.

5. Rinse the piece of tissue in a few changes of distilled water for an hour or more to remove excess osmium tetroxide.

6. To be sure that the solution is removed, rinse overnight under running water.

Then we can put the cut into the cassette and carry out the full process of paraffin infiltration or throw it into formalin for some time. You have to remember that formalin will reduce the osmium tetroxide in the tissue and make it black.

On unstained paraffin sections, all lipids will be black or gray, including triglycerides and lipoproteins.

The mammary gland simultaneously synthesizes several different substances that are released into the follicles. During lactation, the secretory cells produce fat droplets. They combine into larger droplets and are transferred to the region of the apical cells. Eventually, they become part of the vesicular formations in the plasmalemma and are compressed as milk droplets. (2)

Miedź

The copper method for fatty acids is a chemical reaction. Copper forms a salt with a fatty acid and can then be labeled using copper methods such as Howell's rubeanic acid method. The method of salting out fatty acids is not popular today, mainly due to the time it takes to carry out the reaction in the fixed tissue. On the other hand, the methodology of copper dyeing according to Howell:

Methodology

5 µl paraffin sections of tissue fixed in neutral buffered formalin are appropriate. Other fixers are satisfactory.

1. Bring the sections to water with xylene and ethanol.

2. Place in working rubeanic acid solution overnight at 37 ° C.

3. Place in 70% ethanol for 15 minutes.

4. Place in absolute ethanol for 6 hours.

5. Stain slightly with alcoholic eosin as counterstaining.

6. Rinse well with absolute ethanol.

7. Clean with xylene and mount on resin support.

Results:

Copper - red-black granules

Cytoplasm - pink

Comments

• Both negative and positive controls should be used because of the rare staining abnormalities. An appropriate positive control is a liver section from a known case of Wilson's disease. Otherwise, copper can be found in the liver from chronic active hepatitis.

• Copper chelates with rubeanic acid like some other metals. Of these, cobalt and nickel can be confused with copper, so sodium acetate is added to the solution to inhibit them.

• Rubeanic acid is also known as ethanedithioamide, dithioxaamide, or dithioxaline diamide.

The reddish-brown color of the deposits of the copper granules is mainly visible in hepatocytes adjacent to the bile ducts and is mainly perinuclear.(3)

Iron

The colloidal iron technique for acid mucopolysaccharides is similar, iron chemically reacts with the "acid" component of the acid mucopolysaccharides and can then be determined using Perls Prussian Blue, the standard method for iron. In the Schmorl method, the solution is a mixture of potassium ferrocyanide and ferric chloride, which do not react with each other. The reducing substance reduces ferricyanide [Fe (CN) 6] ^3− to ferrocyanide [Fe (CN) 6] ^4−. It reacts immediately with ferric chloride to form Prussian blue, which is insoluble and immediately precipitates where the reducing agent was found.

Both the Perls and Schmorl methods are very common today in the detection of certain cancers, neurodegenerative diseases and chronic liver diseases.

Perls staining showing hemosiderin deposits in proximal tubule cells - "blue kidney" (4)

Staining acc. Schmorl (5)

The question of whether something is impregnation or not, as I mentioned at the very beginning, is quite relative. Due to the fact that metal ions are a component of many dyes, one might be tempted to say that all dyes are impregnation. After all, in most methods, even in H&E, we smuggle iron or aluminum ions so that they show us the appropriate color of the basophilic elements of the cell. On the other hand, detecting metal ions in a cell is nothing more than simply staining or exposing them. So should we be saying on H&E "Hematoxylin impregnation, eosin staining"?

Sources:

1. Liberski, Pawel. (2013). Kuru: A Journey Back in Time from Papua New Guinea to the Neanderthals’ Extinction. Pathogens (Basel, Switzerland). 2. 472-505.

2. Kuehnel, Color Atlas of Cytology, Histology, and Microscopic Anatomy, 4th ed. 2003.

3. 10.Liver (5) Wilson's disease|Pathology Core Pictures

4. Asim, Muhammad & Iqbal, Zafar & Mujeeb, Imaad. (2009). Blue kidney in a pale patient--a case for a causal association between renal haemosiderosis in paroxysmal nocturnal haemoglobinuria and chronic kidney disease. Clinical Kidney Journal. 2. 365-367.

5. Schmorl Melanin Stain Histology Staining Procedure (newcomersupply.com)

6. Baker, John R., (1958) Principles of biological microtechnique Methuen, London, UK.

7. Kiernan, J.A., (1999) Histological and histochemical methods, theory and practice. Ed. 3, Butterworth, Heinemann, Oxford, UK.

8. Bancroft's Theory and Practice of Histological Techniques, 7th Edition (0702042269)

9. DiaPath Perl's stain kit documentation: Perls kit | 010236 | Diapath

10. DiaPath Silver Impregnation kit documentation: Silver Impregnation Stain | Diapath