The histologic Technique includes several procedures
to which a weave is put under to provide the cuts as they are known,
mounted under covers object with images with resisted structures,
for its study under optical or electronic microscopy. For the obtaining
of cuts to observe microscope, it is necessary to follow a protocol
in which the obtaining of the sample, its cut is included and assembly.
1. In order to begin with the histológica technique a sample
of the weave is due to obtain.
2. Fixation: This process talks about to the treatment of the weave
with chemical substances, so that we maintain the cells with the
intact properties best the possible thing. This is obtained when
inactivating certain cellular enzymes that of another way they would
initiate the autolisis and they would take to DEGENERATION POST
MORTEM. The fixation maintains the structures when stimulating the
formation of connections crossed between proteins.
3. Washing: It is made to eliminate the excess of locking device,
so that we soon pruned to make the inclusion without interference
on the part of the locking device. They exist average of inclusion
that is hydrophobic and needs the water elimination in the sample.
We have since to make a dehydration. Because a great part of the
weave is constituted by water, a gradual series of watery solutions
of minor to greater degree of dehydrating agent is applied, for
example, Alcohol Etílico or Acetona. Initiating with alcohol
to 0.5%, soon with a solution of 10%, 20%… 80%, 90%, 95% and
reaching of gradual way the alcohol to 100% to eliminate the water.
This becomes so that if the weave in a solution to the 100% of alcohol
were placed immediately, the water would leave very fast the weave
and it would be become deformed.
4. Explanation or diafanización: After dehydrating the weave,
one goes to a solution of a substance that is miscible as much with
the alcohol as with inclusion means to use (in most of the cases
liquid Paraffin is used like inclusion means). The substance commonly
used is Xylene or Xylol. In the same way the weave sample is placed
in a Xylol container, that single is soluble in alcohol to the 100%.
Explanation is called since the weave becomes is transparent or
clear in xylene, this must to that it changes his refractive index.
Also Tolueno, Benzol or Chloroform like explanation means can be
5. Inclusion: Generally, the weaves are soft and fragile structures,
even after the fixation. Of such form that previous to the obtaining
of the cuts, is necessary to include them in support means. The
average ones more used are the waxes or resins. In liquid state,
these means have the capacity to penetrate and to surround the weave,
of this form can be produced the hardening (by cooling or curing),
to form a solid block that can be cut easily in the microvolume.
Another alternative method exists, that is the fast freezing of
the weave in gelatinous means, that allow the direct tissue cut
of the congealed fragment, that can be cut in immediate form in
a criostato denominated special microvolume. The objective of the
inclusion is to make facilitate the section of the weave to cuts
the sufficiently thin thing like allowing to the passage of the
examined light and being by means of the microscope.
The inclusion is obtained when infiltrating paraffin eliminates
or any means of inclusion in liquid state to the weave, that dissolves
explanation means and penetrates in the weave. Generally the weave
sample is placed in a container and the paraffin fused to 60º
C is added to him, placing the sample in a stove of 30 minutes to
6 hours maintaining the temperature to 60º C. Due to the heat,
the xylol or the benzol evaporates and the spaces previously occupied
by them now are occupied by paraffin. Later the piece is placed
and a little paraffin fused in a mold of paper or metal of rectangular
form and is let solidify to room temperature, forming a solid paraffin
block with the weave piece including, to this block is denominated
to him I mark. The means of inclusion for Electronic Microscopy
commonly used are resins cured, are similar to meth-acrylate.
6. Court: I mark now is possible to be cut in sections the sufficiently
thin thing like allowing the passage of the light. Most of the prepared
ones for optical microscopy have a thickness between 5 to 10 micrometers.
For these cuts a called apparatus MICROTOMO is used, with steel
When we wished to study fats or lipids that are extracted in the
explanation process or enzymes that are inactivated by the heating
of the inclusion, we helped ourselves with the Technique of Weave
Freezing. A congealed weave, is sufficiently it last to be cut.
The liquid Nitrogen weave sample submerges to have a fast freezing.
Soon it is cut with a special apparatus denominated MICROVOLUME
OF FREEZING, exists an elaborated and efficient apparatus more for
the cuts of called freezing CRIOSTATO. The advantage of this technique
is that the cuts that are obtained are very fast, can be used in
I diagnose of pathological material, taken in operations and the
result is obtained in the course of an operation. For Electronic
Microscopy thinner cuts (denominated extreme fine) of approximately
600-800 To of thickness and 0,5 mm of average side are required.
For this a MICROVOLUME WITH LEAF OF GLASS OR DIAMOND is used, that
takes including pocillo made with tape of silver or aluminum. Once
prepared, they mount on copper grids with a diameter of 3mm and
that can be of different forms and material.
7. Assembly: If we want to fix the samples once cut to portaobjetos
to observe optical microscope, we used 2 methods of assembly:
Termostatizado bath: It consists of a bucket within which we were
with the assembly solution, that in our case is formed by gelatin
1% to 38ºC that it makes the function of adhesive of the sample
in the portaobjeto. When we cut to the microvolume, we obtain one
shivers with the cuts, stuck by the ends. This shivers is put floating
on the assembly solution. The cut will be extended slightly, eliminating
the wrinkles and you fold due to the cut, but it will not be gotten
to melt, because the temperature of the gelatin does not reach the
point of fusion of paraffin. Soon, with portaobjetos “we fished”
the samples. This way we obtain in a same porthole several cuts
of he himself block or I mark of inclusion. The portaobjetos take
a cleaning treatment before using to allow better the adhesion of
the cuts. Therefore, water and soap and with water are washed before
with finally distilled. And ether and alcohol to 50% in a bottle
Hot plate: First it is necessary to prepare a assembly solution
(albumen, glycerin.) that works like adhesives. This solution we
will prepare from a solution mother whom there is to dilute. The
portaobjetos we put it on the plate and we added the assembly solution,
drops. On the assembly solution we put the samples to study. We
slipped the excess and we let dry on the plate. In either assembly
methods, the cuts keep in the end in a stove to 38ºC to let
evaporate the assembly solution and finish adhering, like minimum
48 hours. We can leave how long we want before dyeing.
8. Coloration: Indispensable that the samples are transparent or
very clear, and as we will use composed microscope, we must color
or contrast. In order to dye a paraffin cut, previously we eliminated
paraffin because he is insoluble in water, and we do it with reliable
an organic one like tolueno, xylol… In our case desparafinamos
with xylol 3 times 5 minutes every time. Soon we must come to the
hidratación that takes control of a decreasing series of
alcohol. We used first absolute ethanol, soon ethanol to 90%, ethanol
70% and distilled water, 5 minutes every time. And we already can
apply the colored watery solution that yes will be able to dye our
sample. The cuts already hydrated are put in called bottles copleen,
and it is not necessary to never leave dry the samples. After each
coloration it is necessary to make a bath with water. In the case
of the hematoxilina of Harris (color purple) we left 2 minutes it.
Washing does with running water, since turn to the blue one due
to the salts takes place that the water of the faucet contains,
also during 2 minutes. And soon we washed with water distilled during
2 minutes. Soon we dyed with watery eritrosina 1% 2 minutes; we
washed with distilled water 2 minutes. Finally, to put it covers,
previously we dehydrated with ethanol 96% 2 minutes and soon absolute
ethanol 2 times 5 minutes every time. We clarified with xylol 2
times 5 minutes every time. We beat covers with a sticky substance
called eukitt. And we already have it prepared to observe.