EXAMINATION OF FRESH TISSUE Flashcards
1
Q
is the microscopic study of the normal tissues of the body while histopathology is the microscopic study of tissues affected by disease.
A
Histology
2
Q
The procedures adopted for the preparation of material for such studies are known as
A
histologic or histopathologic techniques.
3
Q
The tissues are usually obtained during [?]. They range from very large specimens or whole organs to tiny fragments of tissue.
A
surgery, biopsy, or autopsy.
4
Q
The following surgical procedures are usually performed to obtain the specific-types of tissue that are submitted to a histology laboratory for processing:
A
Fine needle aspiration
core needle biopsy
incisional biopsy
excisional biopsy
Punch biopsy
Shave biopsy
Curettings
5
Q
is the simplest, least invasive test and uses the smallest needle to simply remove cells from the area of abnormality.
A
Fine needle aspiration
6
Q
This is not always adequate to obtain a diagnosis, depending on the area to be biopsied.
A
Fine needle aspiration
7
Q
removes not only cells, but also a small amount of the surrounding tissue.
A
core needle biopsy
8
Q
This provides additional information to assist in the examination of the lesion.
A
core needle biopsy
9
Q
takes out even more surrounding tissue. It takes out some of the abnormality, but not all.
A
incisional biopsy
10
Q
The doctor will slice into the lesion and remove only a portion of it.
A
incisional biopsy
11
Q
If the lesion is found to be cancerous, further surgery may be needed to remove or excise the entire lesion.
A
incisional biopsy
12
Q
generally removes the entire area in question.
A
excisional biopsy
13
Q
is considered the primary technique for obtaining diagnostic full-thickness skin specimens.
A
Punch biopsy
14
Q
It requires basic general surgical and suture-tying skills and is easy to learn.
A
Punch biopsy
15
Q
The technique involves the use of a circular blade that is rotated down through the epidermis and dermis, and into the subcutaneous fat, yielding a 3- to 4- mm cylindrical core of tissue sample.
A
Punch biopsy
16
Q
- where small fragments of tissue are “shaved” from a surface (usually skin).
A
Shave biopsy
17
Q
- where tissue is scooped or spooned to remove tissue or growths from body cavity such as endometrium or cervical canal.
A
Curettings
18
Q
Specimens are usually received in [?] but sometimes they arrive fresh and must be immediately fixed.
A
fixative (preservative)
19
Q
Tissue specimens received in the surgical pathology laboratory should have a request form that lists the [?] along with a description of the site of origin.
A
patient information and clinical history
20
Q
The specimens are accessioned by giving them a [?] that will identify each specimen for each patient. It is important that specimens are properly identified to minimize the risk of [?].
A
number
mislabeling
21
Q
Once tissues are removed from the body, their proteins and cells are digested and broken down by their own enzymes, independent of a bacterial action.
A
autolysis
22
Q
retarded by cold and accelerated at room temperature.
A
autolysis
23
Q
Autolysis is more severe in tissues that are rich in [?] and less rapid in [?].
A
enzymes (e.g. liver, brain, and kidney)
elastic and collagen tissues
24
Q
Methods of tissue examination may vary according to the [?] to be studied, and depends on the [?] to be evaluated.
A
structural and chemical components of the cells
nature and amount of the tissue
25
Fresh tissues are usually examined when there is an immediate need for evaluation. On the other hand, a better and more effective means of studying tissues, whether normal or abnormal, is by examination of adequately preserved sections and smears that are [?] to demonstrate specific structures.
stained
26
The glass slides are then mounted with [?] for permanent keeping.
coverslips
27
Examination may be done on [?], depending on necessity.
fresh or preserved tissues
28
have the advantage of being examined in the living state, thereby allowing protoplasmic activities such as motion, mitosis, and phagocytosis to be observed.
Fresh tissues
29
Its use is limited, however, because of the fact that tissues examined in the fresh state are not permanent, and therefore, are liable to develop the changes that have usually been observed after death.
Fresh tissues
30
Methods of Fresh Tissue Examination
1. Teasing or Dissociation
2. Squash Preparation (Crushing)
3. Smear Preparation
4. Touch Preparation (Impression Smear)
31
3. Smear Preparation
a. Streaking
b. Spreading
c. Pull-Apart
32
is a process whereby a selected tissue specimen is immersed in isotonic salt solution such as normal saline or Ringer’s solution in a petri dish or watch glass, carefully dissected with a needle and separated by direct or zigzag spread using an applicator stick.
Teasing or Dissociation
33
Selected pieces of the tissue are transferred carefully to a microscope slide and mounted as a wet preparation underneath a cover glass, care being taken to avoid forming bubbles.
Teasing or Dissociation
34
It is either stained with a supravital dye or examined unstained by Phase Contrast or Bright Field microscopy.
Teasing or Dissociation
35
It has the advantage of permitting the cells to be examined in the living state.
Teasing or Dissociation
36
The use of the phase contrast microscope greatly increases the structural detail of the cells examined in the living state, allowing movement and mitotic division to be observed.
Teasing or Dissociation
37
The application of certain stains such as methylene blue can be also of great value.
Teasing or Dissociation
38
The preparations, however, are not permanent.
Teasing or Dissociation
39
is a process whereby small pieces of tissue (not more than one mm. in diameter) are placed in a microscopic slide and forcibly compressed with another slide or with a cover glass.
Squash Preparation (Crushing)
40
If necessary, a supravital stain may be placed at the junction of the slide and the cover glass, and allowed to be absorbed by the tissue through capillary attraction.
Squash Preparation (Crushing)
41
The method of preparing the smear differs depending on the nature of the material to be examined.
Smear Preparation
42
As a general rule, smears are made either by spreading the selected portion of the specimen over the surface of the slide with a platinum loop.
Smear Preparation
43
Alternatively, an apposition smear can be made using a second slide to obtain a relatively uniform distribution of secretion.
Smear Preparation
44
Too thin or too thick smears have to be avoided, since they make the tissues less suitable for examination.
Smear Preparation
45
Smears may be examined either as fresh preparations similar to that described for teased preparations, or by using a supravital staining technique.
Smear Preparation
46
Smear preparations can be made permanent by fixing them while still wet, staining them to demonstrate specific structures and inclusions, and mounting the cleared specimen beneath a cover glass with a suitable mounting medium.
Smear Preparation
47
This is useful for preparing smears of thick secretions such as serous fluids, concentrated sputum, enzymatic lavage samples from the gastrointestinal tract, and blood smears.
Smear Preparation
48
This technique is especially useful in cytological examinations, particularly for cancer diagnosis.
Smear Preparation
49
With an applicator stick or a platinum loop, the material is rapidly and gently applied in a direct or zigzag line throughout the slide, attempting to obtain a relatively uniform distribution of secretion.
Streaking
50
Too thin or too thick smears have to be avoided, since they make the tissues unsuitable for examination.
Streaking
51
A selected portion of the material is transferred to a clean slide and gently spread into a moderately thick film by teasing the mucous strands apart with an applicator stick.
Spreading
52
It is a little more tedious than streaking, but has the advantage of maintaining cellular interrelationships of the material to be examined.
Spreading
53
It is especially recommended for smear preparations of fresh sputum and bronchial aspirates, and also for thick mucoid secretions.
Spreading
54
This is done by placing a drop of secretion or sediment upon one slide and facing it to another clean slide.
Pull-Apart
55
The material disperses evenly over the surface of the two slides.
Pull-Apart
56
Slight movement of the two slides in opposite directions may be necessary to initiate the flow of materials.
Pull-Apart
57
The two slides are then pulled apart with a single uninterrupted motion, and the specimen is placed under the microscope for immediate examination, or applied with vital stains.
Pull-Apart
58
This is a special method of smear preparation whereby the surface of a freshly cut piece of tissue is brought into contact and pressed on to the surface of a clean glass slide, allowing the cells to be transferred directly to the slide for examination by Phase Contrast microscopy or staining for light microscopic study.
Touch Preparation (Impression Smear)
59
It has an added advantage in that the cells may be examined without destroying their intercellular relationship.
Touch Preparation (Impression Smear)
60
At times during the performance of surgical procedures, it is necessary to get a rapid diagnosis of a pathologic process.
Frozen Section
61
The surgeon may want to know if the margins of his resection are free from tumor before closing.
Frozen Section
62
An unexpected disease process may be found that requires immediate diagnosis so the surgeon can decide what to do next, or it may be necessary to determine if the appropriate tissue has been obtained for further workup of a disease process.
Frozen Section
63
Immediate diagnosis is accomplished through the use of a [?], especially in intra-operative pathology to help the surgeon in choosing his next plan of action.
Frozen Section
64
It is especially recommended when lipids and nervous tissue elements are to be demonstrated.
Frozen Section
65
Frozen sections are usually done on muscle and nerve biopsies as well as on surgically removed tumors.
Frozen Section
66
A fresh tissue is frozen on a microtome with C02, or on a [?], a cold
chamber kept at an atmospheric temperature of [?].
cryostat
-10° to -20° C
67
The thin [?] are mounted on a glass slide, fixed immediately and briefly in liquid fixative, and stained using similar staining techniques as in traditional wax embedded sections.
Frozen Section
68
For histochemistry, [?] give much faster results than paraffin sections. However, the morphological detail and resolution of frozen sections are usually inferior compared to the quality of tissue that has been embedded in paraffin.
cryostat sections
69
The advantage is rapid processing time with less equipment requirement, and less need for ventilation in the laboratory.
Frozen Section
70
The disadvantage is the relatively poor quality of the final slide.
Frozen Section
71
Frozen sections, both fixed and unfixed, have many applications in histotechnology, and are commonly used for:
1. Rapid pathologic diagnosis during surgery
2. Diagnostic and research enzyme histochemistry
3. Diagnostic and research demonstration of soluble substances such as lipids and carbohydrates
4. Immunofluorescent and immunohistochemical staining
5 . Some specialized silver stains, particularly in neuropathology
72
The tissue for freezing should be fresh, and freezing should be done as quickly as possible. Slow freezing can cause distortion of tissue due to ice crystal artifacts. The more commonly used methods of freezing include:
1. Liquid nitrogen
2. Isopentane cooled by liquid nitrogen
3. Carbon dioxide gas
4. Aerosol sprays
73
is generally used in histochemistry and during intra- operative procedures
Liquid nitrogen
74
most rapid of the commonly available freezing agents.
Liquid nitrogen
75
Its main disadvantage is that soft tissue is liable to crack due to the rapid expansion of the ice within the tissue, producing ice crystals or freeze artifacts.
Liquid nitrogen
76
It also overcools urgent biopsy blocks, causing damage to both block and blade if sectioning is done at -70°C or below.
Liquid nitrogen
77
The tissue snap-frozen in [?] must therefore be allowed to equilibrate to cryostat chamber temperature before sectioning is attempted.
Liquid nitrogen
78
The majority of non-fatty unfixed tissues are sectioned well at temperatures between
-10oC and -25°C.
79
One problem with the use of liquid nitrogen is that it causes a vapor phase to form around the tissue, acting as an insulator that causes uneven cooling of tissue, particularly of muscle biopsies, and making diagnostic interpretation difficult. This problem can be overcome by freezing the tissue in [?] that has a high thermal conductivity.
Isopentane, OCT, or Freon 2.2
80
is liquid at room temperature.
Isopentane
81
is usually suspended in a flask of liquid nitrogen until half-liquid and half-solid stage is reached.
Pyrex glass beaker containing isopentane
82
The beaker is removed from the liquid nitrogen when small crystals start forming on the side of the beaker (approximately [?]), and the tissue to be frozen (affixed on a [?]) is dropped into the cooled liquid isopentane. This is an excellent method for freezing muscle tissue.
-170°C
cork disc, aluminum foil or cryostat chuck
83
Tissue blocks can also be frozen by adapting a conventional freezing microtome gas supply of carbon dioxide gas from a
C02 cylinder.
84
The use of [?] has become increasingly popular in recent years, and is adequate for freezing small pieces of tissue except muscle.
aerosol sprays
85
Quick- freezing spray cans of [?] have a distinct advantage of rapidly freezing blocks of any type of tissue.
fluorinated hydrocarbons (e.g., Cryokwik)
86
Fresh, completely unfixed tissues, or tissues that have been briefly treated with formalin may not require embedding anymore; instead they may be frozen and cut in a [?].
freezing microtome or cryostat
87
Two methods of preparing frozen sections may be resorted to:
1. Cold Knife procedure
2. Cryostat procedure (Cold Microtome)
88
Tissue blocks can be frozen by adapting a conventional freezing microtome gas supply of carbon dioxide gas from a C02 cylinder, or by using a specially made piece of equipment known as cryostat.
Cold Knife Procedure
89
Almost any microtome can be utilized for the purpose, provided means are made available for freezing and maintaining the specimen and the knife at low temperatures, usually by utilizing the carbon dioxide technique.
Cold Knife Procedure
90
A piece of filter paper soaked in gum syrup is placed on the microtome stage, and short bursts of C02 are applied, freezing the filter paper to the stage.
Cold Knife Procedure
91
The selected block of tissue, approximately 3-5 mm. thick, is then oriented on the stage, applied with a few drops of gum syrup and frozen solid with several intermittent bursts of CO2 , each for 1-2 seconds duration, at intervals of around 4 seconds. It should be frozen just to the point where it will be firm enough to section.
Cold Knife Procedure
92
The tissue is then lifted up to the knife manually and trimmed until the surface is flat.
Cold Knife Procedure
93
The surface is then warmed with the finger until the hard frozen tissue starts to thaw and becomes visible to the naked eye.
Cold Knife Procedure
94
This is the DewLine, the point at which sections may then be cut at 10 μm thickness.
Cold Knife Procedure
95
Sections do not form ribbons but rather stick to the knife blade and should, therefore, be removed with a camel hair brush or finger moistened with water.
Cold Knife Procedure
96
They are then transferred to a dish of distilled water to separate, and picked up individually for mounting and staining.
Cold Knife Procedure
97
The water dish is usually placed on a dark or black background, in order to see the sections which are usually colorless or very light in color.
Cold Knife Procedure
98
Tissues that have been frozen too hard will usually chip into fragments when cut.
Cold Knife Procedure
99
The surface of the block may then be softened by warming slightly with the ball of the finger or thumb.
Cold Knife Procedure
100
Tissues that have not been sufficiently frozen will cut thick and crumble, and the block may come away from the stage.
Cold Knife Procedure
101
More bursts of C02 gas should then be given to refreeze the block. Whether used in a cold environment or not, a different temperature between the tissue and the knife is usually employed, the latter being colder.
Cold Knife Procedure
102
Using a cold knife in a controlled cold environment, optimum condition for sectioning shall be provided for by the following temperatures:
Knife -40° to - 60°C
Tissue -5° to - 10°C
Environment 0° to - 10°C
103
The success of this procedure depends upon ambient temperature and humidity.
Cold Knife Procedure
104
It is very hard, if not impossible, to cut sections in a hot or humid environment.
Cold Knife Procedure
105
Ease of cutting and quality of sections are always improved if done in a cold room.
Cold Knife Procedure
106
Sections thinner than 6 μ generally cannot be obtained even from tissues that section well, and with ideal conditions for sectioning.
Cold Knife Procedure
107
an apparatus used in fresh tissue microtomy.
Cryostat
108
consists of an insulated microtome housed in an electrically driven refrigerated chamber and maintained at temperatures near -20°C, where microtome, knife, specimen and atmosphere are kept at the same temperature.
Cryostat
109
The optimum working temperature of cryostat is
-18 to -20°C.
110
Majority of the sections can be cut in isothermic conditions, where the temperature for sectioning can be accurately established and controlled.
Cryostat Procedure
111
The tissue for freezing should be fresh, and freezing should be done as quickly as possible. Slow freezing can cause distortion of tissue due to ice crystal artifacts.
Cryostat Procedure
112
Fresh frozen tissue requires that the tissue be maintained in the frozen solid state during cutting of section, thereby supporting and protecting the tissue from damage and distortion by the knife during the process of cutting.
Cryostat Procedure
113
The tissue must be sufficiently cold to prevent compression and displacement of cell and tissue structures as the knife passes thru it.
Cryostat Procedure
114
The microtome knife needs to be chilled and maintained at low
temperature to prevent complete melting of the tissue, thereby forming a sticky, distorted mass along the knife edge.
Cryostat Procedure
115
When the tissue is too cold, on the other hand, resistance to cutting is increased, so that the tissue becomes brittle and is broken down into fragments upon cutting.
Cryostat Procedure
116
should be left on at all times even when not in use, since it will require several hours to reach operating temperature from a room temperature start.
Cryostat
117
It takes at least one hour for a knife to come down to operating temperature, so that a spare knife should always be kept inside the cryostat cabinet.
Cryostat Procedure
118
To ensure that the sections will cut smoothly and freely onto the knife surface, the knife as well as the undersurface and edge of the anti-roll plate must be kept scrupulously clean and dry.
Cryostat Procedure
119
Soft tissue paper, either dry or moistened with absolute alcohol, may be used to clean the knife and anti-roll plate.
Cryostat Procedure
120
should be defrosted during the weekend, including cleaning and oiling of microtome with special low- temperature oil.
Cryostat
121
The success of fresh tissue sectioning depends to a large extent on the temperature, both of the tissue and of the knife.
Cryostat Procedure
122
Certain tissues such as fat or mucin, and hard or dense structures in a soft matrix require much lower temperatures to impart a suitable consistency for cutting.
Cryostat Procedure
123
These are sectioned on the cryostat by lowering the tissue or knife temperature or both, either by placing the block holder in a bath of alcohol or acetone containing dry ice, or by exposing the tissue to carbon dioxide.
Cryostat Procedure
124
[?] are generally used as mounting media for tissue blocks that need to be sectioned on a cryostat. The O.C.T. (Optimal Cutting Temperature) compound, Lab-Tek Products, Division of Miles Laboratories is especially recommended.
Synthetic water-soluble glycols and resins
125
It is marketed in convenient 8 oz. plastic dispensers in three temperature ranges, depending on the tissue being cut:
[?] for brain, lymph nodes, liver, spleen, uterine curetting, soft cellular tumors;
[?] for non-fatty breast tissue, ovary, prostate, tongue, and GI tract;
[?] for fatty breast and omental tissue.
-5 to -15°C
-15 to -25°C
-35°C
126
The cryostat is usually set at -18 to -20°C.
Mounting of Tissue Block
127
Preferably, the tissue block should be 2-4 mm. thick in order to minimize the risk of the knife hitting the metal tissue block holder.
Mounting of Tissue Block
128
Small fragments of tissue, such as curettings or brain biopsies, are placed on a thick base of O.C.T. compound.
Mounting of Tissue Block
129
The blocks are then surrounded and covered with an additional matrix of O.C.T. compound, and frozen by liquid nitrogen.
Mounting of Tissue Block
130
The frozen tissue is mounted on the microtome.
Mounting of Tissue Block
131
Both the microtome knife and the tissue block are left in the cryostat for 15 minutes at -20°C, to ensure that they are cooled to the correct temperature.
Mounting of Tissue Block
132
Sections between 5-10 μm are then cut slowly and steadily, removed from the knife with a camel hair brush, attached directly to slides of cover-glasses at room temperature, air dried, and fixed (optional).
Mounting of Tissue Block
133
To mount cryostat sections after cutting, one edge of the glass slide is lowered gently until it is about 1/2 to 1 mm. from the knife face.
Mounting of Tissue Block
134
The section will automatically transfer from the cold knife to the relatively warm slide.
Mounting of Tissue Block
135
The slide should never be pressed down on the section, because this will cause a frost mark to remain where the section rested on the knife. If this happens, the frost mark should be wiped away with soft tissue paper.
Mounting of Tissue Block
136
Overall, cryostat sections provide the simplest, quickest and least labor- intensive method for producing frozen sections, and are routinely used for intraoperative and rapid diagnosis of surgical specimen.
Mounting of Tissue Block
137
It should be noted that cryostats cut only individual sections, and do not form ribbons, as in paraffin blocks.
Mounting of Tissue Block
138
Cryostat sections of fresh, unfixed tissue usually attach easily to the slide, even without adhesives, and will preserve enzymes and other substances that may be studied by histochemical techniques.
Freezing Previously Fixed Tissue
139
The cryostat is also recommended for any technique requiring cold sectioning of fixed material, e.g., for fats and lipids, and for some special methods for the nervous system.
Freezing Previously Fixed Tissue
140
Sections of formalin-fixed tissue, however, may not adhere to the slide, and will fall off or be detached during staining.
Freezing Previously Fixed Tissue
141
Clean slides should be coated with albumin or chrome-glycerin jelly so that the fixed tissue will attach to the slide.
Freezing Previously Fixed Tissue
142
Another way, albeit cumbersome, is to immerse the tissue block in boiling 10% buffered formalin for 1 to 2 minutes before freezing and sectioning for rapid surgical diagnosis.
Freezing Previously Fixed Tissue
143
Special fixatives such as 10% formol calcium at 4°C may be used in histochemistry and for lipid demonstration.
Freezing Previously Fixed Tissue
144
Tissues that have been fixed or stored in alcohol should be washed in water for 12-24 hours before sectioning, since alcohol inhibits freezing.
Freezing Previously Fixed Tissue
145
Muscle and nerve biopsies are divided into separate portions that will allow for formalin fixation and paraffin embedding, unfixed snap-frozen for cryostat sections, fixation and resin embedding for electron microscopy (EM) and, in some rare cases, for biochemical immunoblotting studies.
Examination of nerve and muscle
146
Multiple fixation processes are required because multiple techniques are to be used.
Examination of nerve and muscle
147
The portion of a specimen intended for frozen section should be transported on top of wet ice, on saline-dampened gauze, and rapidly frozen within two hours.
Examination of nerve and muscle
148
Upon receipt in the histology lab, specimen is oriented in O.C.T. (Optimal Cutting Temperature) compound and snap-frozen in liquid nitrogen/ isopentane for optimal results.
Examination of nerve and muscle
149
Orientation, size, and expedient flash freezing are critical to obtaining undamaged sections of unfixed muscle fibers.
Examination of nerve and muscle
150
Do not allow the tissue to freeze slowly or to soak up excess saline, as these will cause artifacts that can be seen microscopically and can interfere with diagnostic interpretation.
Examination of nerve and muscle
151
A portion of the tissue is oriented on a piece of cardboard, fixed with 10% buffered formalin and processed for staining with routine Hematoxylin and Eosin (H&E staining).
Examination of nerve and muscle
152
The biopsy portion for electron microscopy is fixed in a buffered solution of glutaraldehyde and postfixed in osmium tetroxide, usually by a specialist in electron microscopy.
Examination of nerve and muscle
153
In some muscular degenerative disorders, biochemical techniques may also be required.
Examination of nerve and muscle
154
For histochemical evaluation involving [?], the tissue needs to be chemically active, and the important chemical constituents should not have been removed, altered or displaced.
enzyme studies
155
is deemed to be the most ideal and preferred means of preserving tissues in order to avoid complete or partial loss of enzymes consequent to chemical fixation.
frozen section
156
Difficulties, however, arise in obtaining thin and serial sections of [?]; since cut sections of tissue tend to disintegrate and cannot be easily handled without prior fixation. These disadvantages will have to be considered in determining the necessity and advisability of such sections.
uniform thickness
157
In addition to fresh frozen tissue sectioning, there are methods that may be resorted to, if chemical fixation of tissue blocks is to be avoided, namely:
1. Freeze-drying
2. Freeze substitution
158
Like fresh frozen sections, these special techniques have the common principle of rapidly preserving the tissue block by [?]. The aim is to produce instant cessation of cellular activity thereby preventing chemical alteration of tissue and displacement of cellular tissue components.
freezing (quenching)
159
must be rapid, accomplished within seconds to prevent the formation of ice crystal artefacts in tissue blocks and produce optimum tissue preservation.
Freezing
160
The freezing agent commonly employed is [?], and the tissue is sectioned into thin slices using a [?] machine under very low temperature.
liquid nitrogen
cryostat
161
The use of [?] and most recently of [?], which can be cooled to very low temperature in order to retain the fluidity of the freezing agents, have contributed much in giving higher conductivity to this liquefied gas.
isopentane, pentane and propane
dichloro-difluoromethane
162
is a special way of preserving tissues by rapid freezing (quenching) of fresh tissue at -160°C and subsequently removing ice water molecules (dessication) by transferring the still frozen tissue block into a vacuum chamber at a higher temperature, e.g. -40°C (sublimation) without the use of any chemical fixative.
Freeze-drying
163
This technique is generally not used in routine surgical laboratories, and is restricted to specialized or research laboratories.
Freeze-Drying
164
A tissue around 2 mm. thick is plunged into isopentane or propane- isopentane mixture which has been chilled to -160° to -180°C with liquid nitrogen.
Freeze-Drying
165
This will effectively solidify the tissue in 2-3 seconds, thus preventing the formation of large ice crystals, autolysis and putrefaction.
Freeze-Drying
166
The frozen tissue is then transferred into a high vacuum drying chamber maintained at a temperature of -30° to -40°C depending upon the size of the tissue.
Freeze-Drying
167
Water is sublimated and dehydrated from the tissue, thereby completing the dessication process within 24-48 hours.
Freeze-Drying
168
Once drying is completed, the tissue is removed, fixed and embedded, either in molten paraffin wax, water soluble waxes or celloidin.
Freeze-Drying
169
Infiltration and impregnation are usually performed in a vacuum embedding oven.
Freeze-Drying
170
The tissue is then sectioned in the usual routine manner and specific staining is applied, depending upon individual necessity.
Freeze-Drying
171
This technique is generally time-consuming and expensive.
Freeze-Drying
172
is by far the most time consuming part of the process, as certain tissues contain 70- 80% water by weight that has to be removed without damage to the tissue.
Drying
173
Furthermore, freeze-dried materials are generally more difficult to section than ordinary paraffin blocks.
Freeze-Drying
174
The tissue is brittle and inadequately supported due to the relatively short period for wax impregnation; hence, it is not advisable as a routine procedure.
Freeze-Drying
175
The tissues are usually flattened directly into an albuminous glass slide with the aid of the finger.
Freeze-Drying
176
Water must be avoided and warm alcohol, acetone, mercury are preferred.
Freeze-Drying
177
Has many outstanding good features.
Freeze-Drying
178
It produces minimum tissue shrinkage, and allows tissues to be processed in a fresh state.
Freeze-Drying
179
It causes minimal chemical change on the cells, most especially on the protein components, and less displacement of tissue and cellular constituents.
Freeze-Drying
180
This method avoids the chemical alteration of cellular components, the denaturation of proteins, destruction of enzymes, and loss of tissue constituents that usually occur in the usual histological processing.
Freeze-Drying
181
This method is particularly important as far as enzyme studies are concerned.
Freeze-Drying
182
In addition to demonstrating hydrolytic enzymes, mucous substances, glycogen and proteins, freeze-drying may be used for special studies, including:
1. Immunocytochemistry
2. Fluorescent antibody studies of polypeptide and polypeptide
hormones
3. Autoradiography
4. Microspectrofluorimetry of autofluorescent substances
5. Formaldehyde-induced fluorescence of biogenic amines (to
demonstrate 5-hydroxytryptamine, adrenaline, and other
catecholamines)
6. Scanning electron microscopy
183
is a process of dehydration, performed at temperatures low enough to avoid the formation of ice crystals and to circumvent the damaging effects observed after ambient-temperature dehydration.
Freeze-Substitution
184
It is similar to freeze-drying in preparing and preserving tissue blocks for subsequent sectioning because both involve the rapid freezing of tissues and the subsequent infiltration and embedding of the frozen tissue block in paraffin or celloidin.
Freeze-Substitution
185
The only variation is that the frozen tissue, instead of being subjected to dehydration in an expensive vacuum drying apparatus, is fixed in Rossman's formula or in 1% Acetone and dehydrated in absolute alcohol.
Freeze-Substitution
186
Infiltration and embedding is then carried out in the same way as in paraffin section.
Freeze-Substitution
187
is based on rapid freezing of tissues followed by solution ("substitution") of ice at temperatures well below 0°C. A 1 mm to 3 mm specimen is thrown into 3:1 propane-isopentane that is super cooled by liquid nitrogen to -175°C (with precautions).
Freeze-Substitution
188
Cryostat sections are cut 8-10 μm, and transferred to water-free acetone (substituting fluid), and cooled to -70oC for 12 hours to 1 week in order to dissolve ice slowly without distorting tissue structure.
Freeze-Substitution
189
The sections are floated onto coverslips or slides and allowed to dry for subsequent histochemical staining.
Freeze-Substitution
190
This technique is relatively more economical and less time-consuming than freeze-drying.
Freeze-Substitution
191
For best morphological and histochemical preservation, substituting fluids should in general contain both chemical fixing agent and solvent for ice, e.g., 1% solutions of osmium tetroxide in acetone, mercuric chloride in ethanol, or picric acid in ethanol.
Freeze-Substitution
