This document provides an overview of histological staining techniques. It discusses how histological preparations are stained using interactions between dyes, solvents, and tissue components. Different staining methods result in different colors that highlight various structures. A classic example is hematoxylin and eosin staining, where hematoxylin stains acidic components blue and eosin stains basic components pink. Specialized staining techniques also exist, such as immunohistochemistry. Proper staining selection depends on the tissue and research goals. Histological preparations are then analyzed under a microscope to study cell and tissue morphology.

1. STAINING AND ANALYSIS of HISTOLOGICAL PREPARATIONS
Introduction to staining of histological preparations
Histological preparations are known in science and medicine for its diverse and
often eye-catching colors. Of course, the primary goal of staining of histological
preparations is not only cosmetic, but need to point out various structural details
of cells and tissues. Given that almost every tissue after cutting on preparations
thickness of only a few micrometers colorless, in most cases it is not possible to
distinguish details on the slide without staining.
Dyeing histological preparations based on different physico-chemical interactions stain,
solvents and tissue components, and these interactions are varied, and depending on the tissue
staining method. These include electrostatic bonds, Van der Waals, hydrogen bonds, covalent
bonds, etc. Stain retention in the tissue is largely dependent on the affinity of the individual
ingredients according to the color of tissue, or affinity of the stain solvent. Key and the
selectivity to the color of tissue components, for which the different structures of different
colors. In addition, the end result will depend on the preparation methods after staining (eg. Of
a substance can be dissolved in alcohol and other organic solvents in the preparation of
dehydration). Many colors in histology are actually neutral salts with acidic or basic radicals.
Because in many cases the color of the affinity for some components of the tissue based on the
interaction of acids and bases. Anions of acid dyes have a high affinity for tissue base
ingredients such as the cytoplasm or proteins in an acidic environment, while the cations of
basic color stain acidic constituents of tissue, such as areas rich nucleic acids (nucleus,
ribosomes, rough endoplasmic reticulum) or sulphated glycosaminoglycans. The tissue was
colored basic color is called basophilic, and one that is afraid of acidic color acidophilus. The
respective colors have affinity for specific tissue ingredients, eg. Natural color resulting from
some lichens, orcein, specifically binds to the elastic fibers and the color shades it a dark brown
color. Anions of acid dyes have a high affinity for tissue base ingredients such as the cytoplasm
or proteins in an acidic environment, while the cations of basic color stain acidic constituents
of tissue, such as areas rich nucleic acids (nucleus, ribosomes, rough endoplasmic reticulum)
or sulphated glycosaminoglycans. The tissue was colored basic color is called basophilic, and
one that is afraid of acidic color acidophilus. The respective colors have affinity for specific
tissue ingredients, eg. Natural color resulting from some lichens, orcein, specifically binds to
the elastic fibers and the color shades it a dark brown color. Anions of acid dyes have a high
affinity for tissue base ingredients such as the cytoplasm or proteins in an acidic
environment, while the cations of basic color stain acidic constituents of tissue, such as areas
rich nucleic acids (nucleus, ribosomes, rough endoplasmic reticulum) or sulphated glyc

2. A special type of tissue interaction and the color is metakromazija. Example
metakromazije staining granules in mast cells in which a high affinity of
the acid radical to the basic granules in aniline dyes (eg. Toluidine) exhibits
the color cluster having a different color than the color of the monomeric
form (violet-red instead of blue). A similar thing can happen in the cartilage
matrix and tissue rich mucins which also has a lot of negatively charged
molecules.
In a transmission electron microscope preparations are not afraid of
conventional stains, but is contrasted by atom of heavy metals (osmium,
tungsten, lead, chromium, etc.) Which strongly scatter electrons, unlike the
atoms composing the biological tissue. There are positive and negative
"staining", depending color you desired structure or its surroundings or
background. Hemalaun-eosin
A classic example of the use of acidic and basic colors in histology staining
technique hemalauneozin, in which use is Hemalaun (base, blue-purple color) and
eosin (acid red-pink color). Acidophilous, eosin colored formations are called
traditional and eosinophilic. Hemalaun colored acidic ingredients tissue dark
shades of blue and purple, for example. The core and rough endoplasmic reticulum,
and the basic ingredients eosin color shades of pink tissue and collagen fibers red
and cytoplasm. If the cytoplasm rich in rough endoplasmic reticulum and free
ribosomes, will be colored shades of purple color due to large amounts of RNA.
Generally, dyeing Hemalaun-eosin gives a good general overview of the structure
of the tissue and therefore the most commonly used method of staining in classical
histology. In the literature one can find the name of hematoxylineosin.
Hematoxylin is a compound which itself is not afraid of tissue, but it is necessary
to oxidize in hematein, who actually colored tissue and stabilize some Mordan
(Stabilizer) and depending on the Mordan, hematoxylin can be divided into several
groups. If such mordan used potassium alum, obtained Hemalaun, the most
commonly used compound hematoxylin in the world. Some other methods that rely
on kiselobaznom interactions are staining Papanicolaou and Romanowski. Some
other staining methods
Although some of the methods used by only one or two colors exist and
Trichrome, tetrachrome and pentachrome staining methods, which use three, four
or five different colors. They are often used when necessary to distinguish the
individual ingredients additional tissue that is not a standard method differ from the

3. surrounding tissue. Some of the known method with three or more colors are:
Trichrome Mallory staining, Masson Trichrome staining, staining tetrachrome
MacNeal pentachrome dyeing method and Movat-Russell. An advantage with more
than two colors are as some different color of muscle and connective tissue, which
is the standard method Hemalaun-eosin predominantly the color eosin pink and red
shades or highlight different types of binder fibers and other ingredients of the
intercellular matrix that would otherwise similar color.
There is also a full range of histochemical methods based on chemical
reactions color and ingredients tissues. These methods allow the determination of
the presence or accommodation for type of molecule in the tissue. These include
methods for detecting the presence of polysaccharides and oligosaccharides, lipids,
various enzymes, ions, and so on. Some of the better known methods of the
PASreaction for the determination of some polysaccharides and related molecules
(in which the reaction is used, and periodic acid Schiff reagent, Eng. periodic
acidSchiff reaction), Prussian blue on the detection deposits of iron in tissues and
Oil red O staining of frozen sections of the lipid.
In histochemical methods include immunohistochemistry and
immunofluorescence, the antibodies which are used in combination with a variety
of labels, such as enzymes and fluorescent molecules. The histology can be used
and fluorescent colors. They stained preparations are examined in a fluorescence
microscope, which is often a source of light, ultraviolet lamp. Fluorescent dye,
illuminated by UV light, emits light of a certain color in the visible spectrum.

4. Special types of staining
The impregnation method is specific for histology at which there is deposition
of heavy metals (silver, gold) on the structures that reduce them from solutions
of their salts. Impregnation and dyeing is not right, because no color is not
included in those structures, but they coat the particles of heavy metals and make
them opaque, but the most common is the simplicity of one of the staining
methods. Impregnation are well displayed bodies of neurons and their cell
extensions, glial cells and reticulin fibers.
To display system tubes or tubules in the tissue, such as the network of
blood vessels, can be prepared injectable preparation. There are various methods
and possibilities for performing this technique, but will method is injection color
or contrast agent into the system vessels or ducts that we want to display before
the fixation of tissue. After the preparation of cuts, tissue, if necessary, can stain
additional contrasting colors.
Bone composition can be hand or mechanically sanded to a very thin bone specimen and soak
in a color which then penetrates into the system and the channel cavities of bones and makes
them visible.
Selection staining methods
The choice of methods is influenced by various factors. Given that there are
hundreds of different methods and their modifications, takes some experience and
basic knowledge of the principles of staining tissue in histology. It is necessary to
understand and that the mechanism behind the staining of tissue specific technique,
as well as the characteristics of the tissue that we want to be afraid. Sometimes,
only one color is enough to show the desired details, for example. Toluidine blue
at rapid pathological diagnosis of malignancy in some frozen sections. Normal

5. staining for histology was Hemalaun-eosin method and it is often used, as in
histological diagnosis, and in order to display the general structure of the tissue,
due to the good contrast between the nucleus and cytoplasm, and display excellent
binding of the fibers. When he wants to highlight the individual components of the
tissue,
Often it is the preparation that precedes staining important if our goal is to stain
the individual ingredients tissues. Eg. the tissue is passed through the organic
solvent which dissolve lipids (such as xylene or one of its alternative procedure
in paraffin) no point in trying to use one of the methods for displaying lipid (Oil
red O or Sudan -black). Similarly, the bone tissue prior to staining in many cases
subjected to a dekalcinacije, in which the inorganic fraction removed from the
tissue. However, if it is planned to use a method for the detection of calcium in
tissues, such as the method by von Kossi, fear must undecalcified bone. In
addition, the possibility of staining is sometimes influenced by the type of
fixative and media for fitting tissues. These examples point out the important
fact that prior research that includes some histological methods mandatory plan
which methods will be used, because it can directly affect the preparation of the
preparation and selection of staining methods. It is good to use streamlined and
standardized protocols and proper controls and consult with people who have
experience with the desired methods. In the end, it is necessary to pay attention
to the supply of quality reagents and adequate storage.
Analysis of histological preparations, morphological analysis
Histological preparation always has a good analysis of the smaller to the larger
increase, but it is good before the microscope to observe and macroscopically to
detect some basic information about the tissue (homogeneity or heterogeneity of
staining, the shape, the structure, the general layout of the tissue or nonuniformity
of its material, the specific structure , the dominant color, larger artifacts, etc.).
Microscopy is usually done in increments of 40, 100 and 400 times, although
increases may optionally be lower (transparent), and the higher (eg. The use of
an immersion lens and a suitable oil). At lower magnifications typically identifies
tissue (if not already known), and analyzes the basic structural details and specific
formation in preparation. Good on a small increase inspect the entire preparation

6. and so are familiar with it and identify the areas that we want to explore in more
detail, as we do in larger increments. When microscopic collect information on
the type, shape, spatial distribution and mutual relations of cells and the
intercellular substances and their quantity, special spaces and cavities, etc. We
analyze the type and intensity of staining and their meaning depending on the
method used.
In light microscopy, most of the preparation has a thickness of 3 to 10
micrometers, which in practice gives two-dimensional display
structures that otherwise have a three-dimensional layout. This should be taken
into account in the analysis of the preparation, but also affects the appearance of
certain components of tissue, depending on their size and spatial distribution. The
direction in which the cut tissue may strongly affect the interpretation of the
preparation, since the individual structures of a very different look in the
transverse, longitudinal and cross section of hair (e.g., muscle fibers, blood
vessels, glands, etc. the outlet ducts.). Therefore, a person who Microscopy
should have knowledge about the structure of tissue observed.
During fixation, fitting, cutting and coloring for various reasons can result in
damage and deformation of the specimen which are called artifacts. Examples
of artifacts are shrinking and separation of the tissue from dehydration, the
dissolution of fat globules and myelin sheaths in xylene, laceration of tissue due
to unevenness microtome blade, uneven thickness of the coloration composition
due to vibration during cutting, stain residue or other chemicals, etc. The artifacts
are to some extent be avoided careful and expert work, but can not be completely
eliminated. Therefore, it is important in the interpretation of the preparation take
account of them.
Quantitative analysis
The science is often necessary to obtain numerical data that can be statistically
analyzed. On histological preparations can be measured and quantified set of

7. values. The quantitative analysis can include many different actions from simple
counting of cells in the visual field to complex stereological measurement and
calculation. For most measurements it is necessary to make a picture of a camera
connected to the microscope and is stored in digital form. If we need data on the
length, area and volume, required calibration system that will allow for the
analysis of digital images we know the relation of pixels and measurement units.
To obtain quantitative data and analysis developed many computer programs,
some of which are paid, are closed source and proprietary companies producing
microscopes and cameras, while some are free, open-source and available to
everyone, while developing them community of scientists and computer
programmers. Some of the basic morphological parameters are the number of
particles, length, volume and area. From them you can get a variety of derived
parameters, depending on the needs of researchers. Since the images are
twodimensional histological preparations, we can not from them directly obtain
three-dimensional data, but with the help of mathematical formulas from
twodimensional measurements can be performed three-dimensional data. Science
that deals with time is called stereology.
It is possible to collect data on the intensity of staining (ie. A semi-quantitative
analysis of immunohistochemical staining intensity), manual or automatic
positioning of certain structures based on color and shape, the separation image
to the channels, to transform the image simpler analysis and 3D reconstruction
of serial tissue sections. Methods for quantitative analysis of the preparation of
the 21st century are more numerous, and their opportunities are growing.
LITERATURE
First Suvarna K, Layton C. Bancroft J. Bancroft's Theory and
Practice of Histological Techniques. 7th Ed. Churchill Livingstone; 2012th
Second Culling CFA. Handbook of Histopathological and Histochemical
Techniques. 3rd Ed. London: Butterworth-Heinemann; In 1974.
Third Durst-Živković B. Practicum histology. 4th revised edition. Zagreb: Mladost;
1998th
4thJunqueira LC, Carneiro J. Basic Histology. 10th ed., Zagreb: Mladost; 2005th
5th Bradbury S. Hewer's Textbook of Histology for Medical Students, 9th ed.
London: William

8. Heinemann Medical Books; In 1973.
6th Xia Y, Momot K. Biophysics and Biochemistry of
Cartilage by NMR and MRI, London: Royal Society of
Chemistry; 2016.
7th Watt IM. The Principles and Practice of Electron Microscopy. 2nd. ed.
Cambridge: Cambridge University Press; In 1997.
8th Bijelić N, T Belovari, Stolnik D, I Lovric, M. Potter Baus histomorphometric
parameters of the
Growth Plate and Trabecular Bone and Wild-Type and Trefoil Factor Family
3 (Tff3) -Deficient Mice Analyzed by Free and Open-Source Software Image
Processing. Microsc Microanal. 2017; 23 (4): 818-825. doi: 10.1017 /
S1431927617000630.
9th Dykstra MJ. Biological Electron Microscopy: Theory, Techniques, and
Troubleshooting. New York:
Plenum Press; In 1992.
remarks
All products and their photos in this material are the property of the Department
of Histology of Medicine in Osijek. Thank Danica Matic, honey. lab. Ing. and
Edi cousin, MSc. Biol. exp. on making preparations.