Connecting and cutting specimens for histological analysis

1. 1
Connecting and cutting specimens for histological analysis
To study the microscopic structure of the tissues and organs, the pattern is to be prepared.
A method of processing sample comprising fixation, inclusion, cutting and staining the sample
sections obtained. Fixation was carried out in order to prevent degradation of the sample due to
autolysis and activity of microorganisms. After fixation is performed fitting, in order to ensure
the necessary strength tissue cutting. Cutting represents the next stage of treatment of the sample.
Using instruments - microtome, samples are cut into thin slices enough that through them can
pass light rays, that can be studied by light microscopy. To study the properties of the tissue using
a microscope (histology) cuts is still necessary to coat.
Thus prepared tissue sample, suitable for the study of microscope is called the
histological preparation. Ideal histological preparation should retain the structure and molecular
composition as in a living organism, but it is in practice difficult to implement. During the
preparation of the preparation sample was subjected to a series of procedures and the various
reagents, however, the individual components of the tissue may be removed (lipid droplets).
When dehydration preparations tissue shrivels, which can lead to the appearance of cavities in
living tissue does not exist. Changes in the tissue as a consequence of tissue processing and
preparation mixture are called artifacts. These changes in the tissue can be reduced to a minimum
by careful handling of the sample and the proper selection of reagents. The quality of histological
preparations depends on each stage of development, and each subsequent procedure depends on
the previous one. Therefore before fixing determining which method will be applied to painting,
The tissues of mammals (mouse, rat, guinea pig, dog, cat, primate) water and fit in a
similar way as human tissues.
When collecting the tissue, tissue mark, in order to avoid a tissue sample during
processing. Such a mistake can ruin the entire previously conducted experiment, whose results

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should be analyzed histologically, or cause dangerous confusion when preparing tissue for
diagnostic purposes. The best way to avoid this is to be labeled tissue cassettes and placed in the
basket for tissue guiding and moved from one set to another reagent.
After fixing, a paraffin prior to incorporation into the resin or plastic, tissue was washed
and dehydrated by the fixative. Due to routine histological fixatives in the art include water (4
and 10% formaldehyde buffered salt solution), the sample was washed with water. In a further
processing stage is required to dehydrate the sample (tissue), for switching said media are not
soluble in water. Dehydration displacement is carried out in samples of higher concentrations
of alcohol (alcohol ascending order). Sensitive tissues (embryonic tissue) begin to dehydrate 50%
of the mixture of alcohol and water, which is valid for large samples. Most tissue samples can
begin to dehydrate 70% alcohol. When the sample is fixed into an alcohol fixative or fixatives
with picric acid (Bouin's fixative), the tissue is directly transferred to the alcohols. From
Bouinovog fixative in 70%, and from alcoholic fixative in 100% alcohol. If the sample
preparation for the analysis of electron microscope after fixation in glutaraldehyde is carried out
in postfixing osmium tetroxide. Dehydration is carried out with the alcohol (or acetone) of lower
concentration (5%) and continues to the 100% alcohol, or with a lower concentration range (10%,
20%, 30%, etc.).
When removed from the sample water, the solvent is moved to fit the media. For the
integration of the paraffin typically used xylene, and such samples after treatment were suitable
for analysis by light microscopy. The sample is moved from the alcohol xylene. When xylene
substitute alcohol in the tissue, it becomes clear, therefore, this process is called enlightening.
For incorporation into the plastic resin used is a solvent for the type of resin, for example.
Propylene oxide. Samples embedded in plastic resin are prepared for analysis by electron
microscopy, and in some cases can be analyzed by light microscopy (Semi-thin sections).

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Sampling
Incorporation of samples comprising sample fluid permeation switches and formation of
blocks. Paraffin is the most commonly used medium for the switching of light microscopy. It is
a mixture of chain hydrocarbons in the form of a solid, whitish mass odorless and Supplied in
the form of leaves or grains. It is chemically inert. Melting point is between 45-60 ° C (usually
used paraffin with a melting point between 56-58 ° C). The liquid paraffin has a cavity in a
sample, when cooled rapidly solidified. Allows to obtain high quality cuts and is suitable for
most routine and special staining, including immunohistochemical method. Below will be
described the process of incorporating in paraffin and cutting paraffin blocks.
The paraffinic process, permeation takes place in a thermostat at a temperature of melting
point paraffin wax. The three vessels in the thermostat of the dam liquid paraffin. Paraffin volume
in the vessel to be 25-30 times the volume of the sample. The first vessel was added, and the
sample every two hours the sample is transferred into the next container. During permeation,
Paraffin replace xylene in the tissue voids. If tissue is not well dehydrated and infiltrated xylene,
or is not completely saturated with paraffin, when cutting is broken and crumbling.
Designblocks takes place outside the thermostat. The sample from the last container with
paraffin in a thermostat filled mold moves into the liquid paraffin. If it is manually can use
different molds for shaping blocks. L-molds consisting of two metal parts of the L-shaped, whose
stacking may form smaller and larger molds, depending on the sample size. Molding blocks
should be so large that some tissue remains sufficient amount of media integration that will
provide support tissue during cutting. When preparing L-molds, metal parts are assembled on a
metal or glass plate, and when the wax cools easily removed. If you use glass molds, previously
it is smeared with glycerine. The process is carried out so that the mold of the dam liquid paraffin,
and then the sample is placed with tweezers in the middle of the mold. In this tissue landmarks,
a block mark. Properly orient the sample mean cutting surface facing the bottom of the mold and
set in parallel with the substrate, in order to receive the cut when cutting a selected part of the
sample, and the cut affecting the entire surface. Incorrect orientation of the sample may impede
the microscopic analysis of the sample.

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The entire process should be fast in order to avoid cooling of the mold in paraffin and paraffin
wax in the sample, because then a layer of solidified paraffin around the sample. Therefore, the
tissue does not connect well with paraffin in the mold and when cutting the pattern is separated
from the rest of paraffin. After being placed in the mold, the label under which is then driven to
the fixed to the paraffin block. The paraffin blocks were then placed on cooling. As paraffin gets
colder, the paraffin crystals which occur in this process are smaller.
Most laboratories today has a greater or lesser extent automated sample processing.
Automatic tissue processors are programmed so that samples of the tapes are transferred from
one agent to another for 24 hours, and in them, the tissue can be performed by fixation to the
permeation of paraffin. To format the blocks there are appliances in which liquid paraffin is
poured into molds, but orientation samples employees work in the laboratory. Cutting is still
carried out manually. Staining can also be automated.
Besides paraffin there are other media integration. They are applied when reagents in
paraffin is removed from or damage the components of tissue which are to study (lipids), the
energy required for switching in paraffin affect tissue or enzymes, the medium for incorporation
is not firm enough to tissue or required thinner sections than are get cutting paraffin blocks. In
this case the synthetic resin. Is used for switching of hard tissues such as bone and uterus, or
special preparation (about) celloidin used because it provides better support tissues. In addition
to fitting in celloidin not require heating. Disadvantages cellodina (longer time required for
making preparations, difficult getting cuts thinner than 10μm) and adjustment properties of
paraffin by adding various additives have led to today celloidin rarely used. In addition to the
aforementioned means which are not soluble in water, and the media are water soluble (eg.
Carbovax, gelatin, agar).

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Frozen cuts
A different method of preparing a composition, which can avoid the influence of heat on
the reagent and the sample preparation of frozen sections. With this method, the sample is rapidly
frozen, and so at the same time fixes and hardens. Frozen samples are cut using a cryostat,
sections are mounted on glass and further processed. It is suitable for quickly getting cuts and
different histochemical research.
Incorporation of samples for transmission electron microscopy
For the study of the ultrastructure of tissue cells and an electron microscope is used. In a
transmission electron microscope, electrons passing through the sample, but the cuts a much
slimmer than for light microscopy. Therefore, using special funds for integration. The most
commonly used resin for switching the group of epoxy resin (Epon and its replacement). Most
of the epoxy resin is a transparent yellow color. As well as for light microscopy, and electron
microscopy sample after fixing dehydrated. If the spent dehydrating the alcohol, and the sample
fits into the plastic resin prior to incorporation was immersed in the solvent
of the resin, of propylene oxide. Embedding tissue is performed so that the solvent concentration
of the resin gradually decreases, and the concentration of media for inclusion increases. When
the shaped blocks, in capsules or for incorporation into molds receive media switches and the
sample and allowed to polymerize the resin. EPON is polymerizable by heat. The polymerization
by means of heat is performed by heating the medium to fit to the sample (in the mold) at a
temperature between 35-60 ° C. Some resins can be other than heat, and UV-cure.
Cutting samples
Following the procedure in the development of histological preparations is cutting. For
light microscopy analysis, samples may be cut into sections the thickness of 1-10 um (microns 1

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is 10- 6 m) by using a special apparatus - microtome. The thickness of the cut is different for
different types of tissue (glandular tissue is a regular slot on thinner and brain into thick slices).
Cut samples of paraffin embedded
Code rotary microtome with a pattern block is fixed to the movable holder, a knife is
mounted to the holder which is stationary. The block is adjusted such that is parallel to the knife,
the knife and the sample is closer to the device several thin sections to remove the paraffin
surface. Turning the handle of the tissue is moved from up to down, and the pattern block slides
over the blade and thus the cut section. When the carrier completes movements, lever or a
complete revolution with the tissue carrier is automatically moved to the knife so much
micrometers is set slice thickness. Rotary microtomes are suitable for cutting paraffin embedded
tissue, especially soft tissue, and for serial sections. sliding microtome a moveable blade holder,
and the block with the tissue is fixed to a holder which is movable. Pulling the knife slides over
the sample block and in that the cut section of a particular thickness. Sliding microtomes are
suitable for cutting soft and hard tissue (whether embedded in paraffin or celoidin) and for cutting
of larger samples (such as around).
When cutting paraffin block using a rotary microtome, the block glides over the knife
blade and thereby cut cut that remains on the knife. Wet brushes may each cut off knife and
transferred to a dark background, or can be set to cut the block. Then the paraffin paste the cut
edge prior to the edge in paraffin following the cut, the strips form a paraffin. When creating
serial cuts, all the cuts collect and properly, following the cutting, agree on a dark background.
The resulting cuts to the naked eye look like whitish translucent leaves. In the following
stages of processing the sample to be at a dark samples to be easily viewable. Because of the
pressure in the stroke accumulated paraffin and so cuts obtained after cutting plane.
Handling paraffin sections is carried on the surface of hot water in a water bath or on a heating
plate. When the sections equal to the surface of hot water in a water bath of distilled water was
warmed to a temperature a few degrees lower than the melting point of paraffin. Cuts to brush
transmitted to the surface water. Heat yield and paraffin sections are smoothed. Then the slide
angled plunge into the water below the cut and cut gently pull on the glass. If the flat liquid wax
strips, when placed on the glass cuts apart and each mounted on a glass. When straightening
sections on the hot plate, the glass slides and placed on the water surface of the water is a section
or several sections in the form of stripes of paraffin. Then the glass with slices put on the hotplate.

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Heating the water, is heated and stretched paraffin and which leads to the equalization sections.
If necessary it can be histological needles or brush additionally compensated, but it needs to be
carefully done to avoid tearing the cut. After that, the slide with the cuts is removed with hot
plates.
Mounting cuts a process wherein the flattened sections are placed on glass slides,
individually and in series. The method is carried out upon handling, while the glass is still water,
which allows movement of the glass cutting. Placed in the middle of the glass, and at the same
time can be further corrected and orient. Thereafter, excess water wiped the glass with slices are
placed on the hotplate that cuts dry and solidify on the surface of the glass. Depending on the
following procedure in the development of histological preparations selected slides to which will
be mounted cuts. For different histological staining sections are mounted on plain glass slides.
Should be implemented immunohistochemical method, the incisions are mounted on adhesive
glass. Mounting adhesive glass is recommended for cuts decalcificated bones, to avoid the
separation of the sections of the glass in the following process of the sample processing. In the
method of cutting a sample and mounting the bit cuts a tag, on which the pattern is then guided
to, rewrite to the glass slide.
Serial cuts are made when analyzing the structure of complex three-dimensional structure
or analyze samples in which components constituting not represented in the whole sample.
Histological analysis of a cut, the usual thickness of 6 microns provides limited insight into the
structure of the three-dimensional scaffold. However, analysis of shearing sections mounted on
glass can be monitored structure or shape across and study a structure of the sample. Serial
sections are prepared so that the whole sample is cut away and keep all cuts. Collected sections,
from first to last row is agreed as a paraffinic ribbons on a dark background. Then, in that order
governed and mounted on glass. If it cuts a lot, then during the celebration of glass, with a label
under which the sample conducted required Enter the number of the glass.

8. 8
Dewaxing and rehydrating
After mounting on the glass, paraffin-embedded sections are to be prepared for the next
stage of treatment of the sample - dyeing. From the cut first remove paraffin and then the tissue
returns water. A method of removing paraffin from the cut is called the dewaxing. Is carried out
by dipping sections in xylene or other solvent for the paraffin. Due to its harmful effects, in recent
years increasingly used replacements for xylene. Since the colors that are used during the
processing of the sample for histological analysis, mostly aqueous solution of complex organic
compounds, prior to staining the tissue need not leave the water. This process is called rehydrate.
Carried out after the dewaxing, dipping sections in all lower alcohol concentration (alcohol
downward range, from 100% to 70% alcohol) to the water. For the analysis of light microscopy,
samples can be painted different colors histological. Color selection is dependent on the tissue,
whether he intends to be a prominent ingredients in the tissue for analysis, and the preceding
process in the processing sample.
Cutting specimens embedded in resin
Samples of the blended resin, the electron microscopic analysis, the cut ultramikrotomom.
This is a special type of microtome which allows cutting thin slices. Cutting using glass or
diamond blades. When cutting block patterned glides over the knife and cut off the cuts are
collected on the surface of the water behind the blade. Thickness of thin cuts
is between 0.1 to 2.5 mm, but the majority is between 0.5 to 2 microns. The cut sections are
transferred to a drop of water on glass, dried on a heating plate (at a temperature 70 - 80 ° C) and
stained with toluidine blue. The sections (compositions) suitable for the analysis of tissue by light
microscopy, for diagnostic and experimental purposes, the sample quality assessment and browse
to select sample areas to be subjected to a further process. The selected part of the sample is then
cut into the even thinner sections ( ultra-thin cuts) suitable for electron microscopic analysis.
Thickness of ultra thin sections is between 8-100 nm. The preferred thickness of the ultra thin
sections approximately 80 nm (1 nm-10 9 m).

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Ultrathin sections which are collected from the surface of the water behind the blade of
the knife, mounted on the mesh. Nets are usually made of copper, but can be made from other
materials such as nickel or gold. The outer diameter of the mesh is 3 mm. The sample mesh
openings in a variety of information in a grid (mesh), the hexagon as shown; or in the form of
parallel stripes. In a further sample preparation for analysis by electron microscopy, sections
mounted on the mesh contrasting salts of heavy metals.
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2007th
4th Hayat MA. Principles and techniques of electron microscopy. Biological
applications. 4th Ed. Cambridge: Cambridge University Press; 2000th 5th
Junqueira LC, Carneiro J. Basic Histology. 10th ed., Zagreb: Mladost; 2005th
6th Prophet ED, Mills B Arrington JB, Sobin LH. Laboratory Methods in Histotechnology.
Washington, DC: American Registry of Pathology; In 1994.
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