PREPARED BY: PROF.DR. NAHED AHMED OMAR

(Histochemistry &Microtechnique) For 2nd students

Zool./Chem. Credit Hours

PREPARED BY:

PROF.DR. NAHED

AHMED OMAR

Micro technique :is the preparation and

staining of tissue sections from surgical

or autopsy material for microscopic

study.

Cytology : is the study of individual cell

microscopically.

Histology: is the study of specialized cell

aggregates from definite structures with

specific function.

Histological technique deals with the

preparation of tissue for microscopic

examination. The aim of good histological

technique to preserve microscopic anatomy

of tissue and make them hard, so that very

thin section (4 to 5 micron)(1000 micron = 1

millimeter) can be made. After staining, the

section should represent the anatomy of

the tissue as close to as possible to their

structure in life. This is achieved by passing

the total as selected part of the tissue

through a series of process.

What happens to the organism when they die?

When an organism dies, the tissues and cells

degrade and disintegrate due to two factors: -

A. Bacterial disintegration

B. B. Self-decomposition (Autolysis)

Why do the fixation process or what are the advantages of the fixation?

This process in order to achieve the following: - Prevent decay or decomposition of the self- bacteria Survival components its natural state before fixation Convert dissolved material into insoluble materials Make tissue access coloring dyes What is fixative? Fixative is a chemical permeates the tissue to fixed its contents and kept on their natural state before fixation.

These processes are:

1. Fixation

2. Dehydration

3. Clearing

4. Embedding

5. Cutting

6. Staining

Before start of this process we making prefixation(Washing) with saline solution.

Fixation is usually the first stage in a multistep process to prepare a sample of biological

or other analysis. microscopymaterial for Therefore, the choice of fixative and fixation protocol may depend on the additional processing steps and final analyses that are planned. For example, immunohistochemistry uses antibodies that bind to a specific protein target. Prolonged fixation can chemically mask these targets and prevent antibody binding. In these cases, a 'quick fix' method using cold

hours is typically used.24 for around formalin

Fixation: is the process by which the constituents of

cells and tissue are fixed in a physical and partly also in a chemical state so that they will withstand subsequent treatment with various reagents with minimum loss of rchitecture. This is achieved by exposing the tissue to chemical compounds, call fixatives.

biological is a chemical process by which fixation

are preserved from decay, either through tissuesautolysis or putrefaction. Fixation terminates any ongoing biochemical reactions, and may also increase the mechanical strength or stability of the treated tissues. Fixation preserves a sample of biological material

(tissue or cells) as close to its natural state as possible in the process of preparing tissue for examination

1- Confers chemical stability on the tissue

2- Hardens the tissue (helps further

handling)

3- prevent enzyme autolysis

4- Prevent bacterial putrefaction

5- May enhance later staining techniques

6- protect the cells from distortion and

shrinkage when they are subjected to

alcohols and hot paraffin.

How fixative interacts with the contents of

tissue?

Interacting functional groups in the fixative

with functional groups of chemicals to

fixed by forming a links as Cross linkages

or Bridge linkages between them.

Examples functional groups: -

Carboxylic groups COOH

Hydroxyl groups OH

Groups Aldehyd CHO

Amino groups NH2

Groups sulfur SS

SH Hydrogen sulfate groups

Mechanism of action of fixatives:

Most fixatives act by denaturing or

precipitating proteins which then form a

sponge or meshwork, tending to hold

the other constituents.

Good fixative is most important factors in

the production of satisfactory results in

histopathology. Following factors are

important:

• Fresh tissue

• Proper penetration of tissue by fixatives

• Correct choice of fixatives

No fixative will penetrate a piece of tissue thicker than 1 cm. For dealing with specimen thicker

than this, following methods are recommended:

1. Solid organ: Cut slices as necessary as but not thicker than 5 mm.

2. Hollow organ: Either open or fill with fixative or pack lightly with wool soaked in fixative.

3. Large specimen, which require dissection: Inject fixative along the vessels or bronchi as in case of lung so that it reaches all parts of the organs.

A. Tissue fixatives a. Buffered formalin b. Buffered gluteraldehyde c. Zenker’s formal saline d. Bowen’s fluid B. Cytological fixatives a. Ethanol b. Methanol c. Ether C. Histochemical fixatives a. Formal saline b. Cold acetone c. Absolute alcohol

By far the most commonly used fixative in histology . It is usually used as formaldehydeis

10% Neutral Buffered Formalin (NBF), that is aprox. . phosphate buffered saline% formaldehyde in 3.7

Because formaldehyde is a gas at room temperature, formalin=formaldehyde gas dissolved in water (~37% w/v)-is used when making the former fixative. Routine Formalin: Formalin is sold as 40% w/w solution of formaldehyde

gas in water. It is used as 10% solution in water or normal saline. It does not precipitate protein but combine with NH2 group to form an insoluble gel, preserve particularly all elements including fats. It keeps phospholipids insoluble in fat solvents. Tissue can remain in it for prolonged periods without distortion. It is compatible

with most special stain. It is the cheapest and most popular fixative.

. It glutaraldehydefor fixation is aldehydeAnother popular

operates in a similar way to formaldehyde by causing

deformation of the alpha-helix structures in proteins.

However glutaraldehyde is a larger molecule, and so its rate

of diffusion across membranes is slower than formaldehyde.

Consequently glutaraldehyde fixation on thicker tissue

samples may be hampered, but this problem can be

overcome by reducing the size of the tissue sample. One of

the advantages of glutaraldehyde fixation is that it may

offer a more rigid or tightly linked fixed product—its greater

length and two aldehyde groups allow it to 'bridge' and link

more distant pairs of protein molecules. It causes rapid and

irreversible changes, fixes quickly, is well suited for

electron microscopy, fixes well at 4oC, and gives best

overall cytoplasmic and nuclear detail. However it is not

ideal for immunohistochemistry staining.

Some fixation protocols call for a combination of

formaldehyde and glutaraldehyde so that their respective

strengths complement one another.

Washing tissue after fixation:-

1-After the tissue is fixed for the proper length of

time, excess fixative must be washed out to

prevent over-fixation. Washing also removes

substances in the fixative which might interfere

with the subsequent processing.

2- Since most fixatives are aqueous solutions, the

washing is usually carried out for a specific

period of time in tap water or isotonic saline

solution.

Tissue Processing

Tissue processing is a long procedure and required 24 hours. Tissue processing can be done by manually or mechanically.

Manual Tissue Processing: In this process the tissue is changed from one

container of reagent to another by hand. Mechanical Tissue Processing: Automatic tissue processors are available. In this

processor, there are different jars containing reagents. These are arranged in a sequence. The tissue is moved from one jar to another by a mechanical device. Timings are controlled by a timer which can be adjusted in respect to hours and minutes. Temperature is maintained around 60°C.

The processing, whether manually or mechanically, involves the same steps.

Dehydration

Tissues are dehydrated by using increasing strength of alcohol; e.g.

50%, 70%, 90% and 100%. The duration for which tissues are kept

in each strength of alcohol depends upon

1.the size of tissue,

2. fixative used

3. type of tissue

to be dehydrated slowly starting in 30% ethyl alcohol directly

whereas most tissue specimens may be put into 70% alcohol.

Delicate tissue will get high degree of shrinkage by two great

concentration of alcohol.

The volume of alcohol should be 50-100 times that of tissue

1.Tissues contain much "free" water which does not mix well with

the paraffin used later in the procedure. Therefore, water in the

tissues must be removed by submerging the tissue in alcohol, a

process known as alcohol dehydration.

2.If this is done too rapidly, the large out flow of water can damage

the morphology of the cells and tissues.

3.In this step, tissue is placed into a series of gradually increasing

concentrations of alcohol, usually ethyl alcohol (30, 50, 70 ,80, 95,

and 100%), for specific periods of time.

Dehydrating agents

1.Alcohol

Advantages of alcohol:-

They are fast acting

Non toxic

Reliable

Clearing

(Removal of Alcohol)

During dehydration water in tissue has

been replaced by alcohol. The next step

alcohol should be replaced by paraffin

wax. As paraffin wax is not alcohol

soluble, we replace alcohol with a

substance in which wax is soluble. This

step is call clearing.

The following reagents of clearing are:-

• Xylene

• Chloroform

• Benzene

• Carbon tetrachloride

• Toluene

Impregnation with Wax

( infiltration )

This is allowed to occur at melting point temperature of paraffin wax, which is 54-60oC. Volume of wax should be about 25-30 times the volume of tissues. The duration of impregnation depends on size and types of tissues and the clearing agents employed. Longer periods are required for larger pieces and also for harder tissue like bones and skin as compared to liver kidney, spleen,lung etc. Xylene is easiest way to remove. Total duration of 4 hours is sufficient for routine impregnation.

Infiltration: is to impregnate the tissue

with supporting medium to facilitate its

cutting by microtome knife.

Types of Wax employed for

Impregnation:-

1. Paraffin wax

2. Water soluble wax

3. Other material, like colloidin, gelatin,

paraplast etc.

Embedding

(Blocking )

Impregnated tissues are placed in a mould

with their labels and then fresh melted wax is

poured in it and allowed to settle and solidify.

Once the block has cooled sufficiently to form

a surface skin it should be immersed in cold

water to cool it rapidly.

After the block has completely cooled it is cut

into individual blocks and each is trimmed.

Labels are made to adhere on the surface of

the block by melting the wax with a metal

strips sufficiently wormed

Embedding media: are all materials used

by histological technique to infiltrate ,

support and enclose specimens which

are to be cut into thin sections by a

device or instrument called microtome.

The paraffin used in histology laboratory is a refined , white , filtered paraffin , to which has been added : 1. beeswax and Paraffin wax 2. Paraplast 3. Paraplast plus 4. Gelatin 5. Celloidin , to facilitate riboning:

Paraffin is sold at room temperature . Heat renders paraffin fluid , so that it can permeate the tissue. Hardness of paraffin used for infiltration is matched to the hardness of the tissue. Cakes of paraffin are placed in clean metal or enamel pitchers and melted down in a paraffin oven regulated at a temperature just above the melting point of paraffin. The melted paraffin is filtered within the oven by coarse filtered paper and is then ready to use ( to reduce injury to the knife edge when sectioning the tissue.

Blocking , Orientation of specimen ,

Trimming and Cutting

Microtome

Staining

1- keep stains and solutions covered when not in use

2- filter stains before use 3- after the slides are removed from the drying

oven, allow them to come to room temperature before placing in xylene (dewax).

4- once the slides have been placed in first xylene to remove the paraffin, do not allow them to dry out.

5- Make certain that the level of any solution used in staining, completely covers the tissue on the slide

6- Re-new water bath after each rack of slides that has been processed .

7- Drain all slides before removing on to the next solution.

8- Use the microscope for quality control.

Staining methods may be grouped as

follows:

1- Vital staining

2- Routine staining

3- Special staining

Every stain is to be used according to a specified method. Staining can be done either manually or in an automatic stained.

Manual Staining:

In a small laboratory when a few slides are stained daily, this is the method of choice. Although it is time consuming it is economical. Different reagent containers are placed in a special sequence and the slides are removed from one container to another manually.

Automatic staining:

Step time

Dewax in xylene to remove wax from slides

Hydration by series alcohols then bring sections down to water

Hematoxylin solution. rinse

Wash in running water 3 min.

Eosin rinse

D.W

Dehydrate in ethanol and clear with xylene

Mount in DPX or Canada balsam

Definition Histochemistry : Is the science that deals with identifying the chemical components of the animal tissues and determine their locations in the sites by using chemical methods. What does the term mean Histochemeia: This word is derived from the Greek origins Histos = tissue Chemeia = transmutation

We say specialized Histochemistry =

Histochemist

Histochemistry is the study of the chemical identification and distribution of

within and between biological compoundscells using histological techniques such as

(optical) light, indicators and histology stainsand electron microscopy.

Histochemistry is the aspect of histology concerned with the identification of chemical components in biological cells and tissues. So, whereas histology in general is the study of biological cells and tissues in fine microscopic detail using special histological techniques, histochemistry is concerned specifically with the chemicals within, between, and forming the biological cells and tissues themselves.

Histochemistry is the identification and

study of chemicals within and around

the microstructure of biological cells and

tissues observed using

techniques to prepare particular

and microscopes histological specimens

to observe those specimens.

Tissue consists of several essentials components

behind water, namely : -

1- Inorganic substances, Essential and Non essential

2- Organic substances Nucleic acids Carbohydrates

Proteins Lipids Pigments

The difference between the science of histochemistry and Histology:

- Histology is interested in studying the morphological structure components of the cells and tissues as it seen under the light and electronic microscopes.

- Histology is the scientific study of the fine detail of

biological cells and tissues using microscopes to look at specimens of tissues that have been carefully prepared using special processes called "histological techniques".

- While histochemistry is interested by studying chemical components of tissue and identify their locations and identify them. What is the importance of histochemistry : Because histochemistry serves a lot of science and perhaps the most important is the histopathology.

Uses of Histology

- Education - Histology slides are often used in teaching laboratories to help

biological students learn about the microstructures of human (and animal)

.tissues

- Diagnosis for treatment - Biological tissue samples taken from a patient

(that is, a specific person or animal's body) may be studied in detail to

enable medical or veterinary experts to learn more about the patient's

condition and hence perhaps understand its causes and make

recommendations for treatment or management of the condition.

Note: Although the study of the microstructure of diseased cells and

tissues (e.g. to help inform decisions about treatment in clinical medicine)

is an aspect or use of histology because it uses histological techniques,

.histopathology study of diseased tissues is more accurately called

- Forensic investigations - Forensic histology, immunohistochemistry and

cytology involving microscopic study of biological tissues using various

stains can help clarify the cause of sudden unexpected deaths and other

issues in forensic science.

- Autopsy - Biological tissues from a deceased person or animal can be

studied using histological techniques enabling experts (e.g. pathologists re.

unexplained death of a person) to learn about the circumstances and

possibly cause of death.

- Archaeology - Study of biological cells and tissues recovered from

archaeological sites can provide information about history, even ancient

history. The state of preservation of the biological material is critical and

sometimes sufficient e.g. for bone histology and dental histology.

Special staining : special or selective

staining demonstrate special features of

the tissue , such as protein ,carbohydrate

or particular cell products . e.g. Fulgen

stain for DNA

Special stains This is the bases of histochemistry. in which the

identification of certain structures and chemical substances is accomplished by specific chemical reactions designed to give a final color at the site or location of the structure or substance in the cell or tissue , such specific stains give little or no affinity for other tissue elements .

Examples of specific stains are the demonstration of DNA with Feulgin's technique,

hemsiderin with Parl's Prussian blue reaction , polysaccharides with Periodic Acid Schiff

technique(PAS). 1.Van Gieson's stain for collagen fibers This is a very useful and simple stain that may be

employed after any fixation.

Elastic tissue fibers - Verhoeff's Van Gieson stain(EVG):-

This stain is useful in demonstrating atrophy of elastic tissue in cases of emphysema, and the thinning and loss of elastic fibers in arteriosclerosis, and other vascular diseases. With increasing age, changes such as splitting and fragmentation occur, these changes are most obvious in the skin which becomes wrinkled

Best’s carmine stain for glycogen:-

Glycogen is a simple intracellular

polysaccharide which exists as the

storage form of glucose . It is found in

abundance in liver , muscles (skeletal

and cardiac) and epithelium of cervix

and vagina.

Good fixative for glycogen must be

alcohol based ( glycogen is soluble in

water but not in alcohol)

Periodic acid Schiff reaction (PAS) for

Mucin and Glycogen:-

Purpose: Glycogen is present in skin,

liver, parathyroid glands and skeletal

and cardiac muscle. The PAS stain is

used for demonstration of basement

membranes, fungus secreting

adenocarcinoma from undifferentiated

squamous cell carcinoma, and muco-

substances secreted from the epithelia

of various organs. A routine stain for

liver and kidney biopsies.

Congo Red for amyloid:-

Purpoes: To demonstrate amyloid

deposits in tissue sections

Principle: Amyloid is an extracellular

abnormal protein complex deposited in

various tissues , particularly in and

around blood vessels in certain disease

status, it may cause damage to the

surrounding tissues.

Gomori's iron reaction for iron pigments identification:-

In this reaction , the loosely bound ferric iron in the tissue combine with potassium ferrocyanide to form ferric – ferrocyanide which has a bright blue color.

Diagnostically : this method can detect small amount of Fe+++ normally found in hemosiderin ( blood pigment) , spleen , bone marrow, and in macrophage ( heart failure cells) within lymph glands and lung alveolar spaces.

Purpose : demonstration of iron pigment in tissue .

Decalcification of

Bone

Preparation of bone sections

Bone require special treatment , because of its hard

nature . before bone or any calcified tissue can be

processed and sectioned by microtome , it must have the

calcium salts removed .This process is called

(Decalcification) .

Removal of calcium is achieved with acids in which

the calcium carbonate and phosphate salt of the bone

are soluble.

The time taken for complete decalcification is dependent

on :-

1. Thickness of the specimen

2.Compactness or density of the bone .

3.Strength of the solution used .

4.The temperature at which it is decalcified. ( higher than

room temperature ,accelerate decalcification , but

impair staining) .

Decalcification above 50 C cause disintegration . Bone specimen must be cut into fairly small pieces first . A, Very dense and hard bone 2-5 mm. thick. B. Softer tissue 4-6 mm. thick , to allow penetration of the decalcifying solution . A saw with thin blade should be used , it produces less tearing of the surrounded tissue. As soon as the small sections are obtained , the bone must be fixed to preserve and toughen the soft tissue and cellular structures attached to it . buffered 10% neutral formalin is recommended for use as a fixative for bone because it penetrate well and render the soft tissue resistant to the acids present in the decalcifying fluid. After fixation , the tissue must be washed thoroughly to remove the excess fixative . It is then ready for decalcification.

Methods of decalcification 1.Acid method

2.Ion – exchanging resins 3.Electrical ionization. 4.chelating method.

Acid method The acid method is the most widely used for routine processing of large amounts of bony tissue. Decalcification of the fixed , washed tissue should be carried out at room temperature in large jars with decalcifying solution added generously . The agent most used for decalcification are aqueous solutions of nitric acid , formic acid and tri-chloro-acetic acid. The ideal time for decalcification of tissue is 24-48 hrs. Some time , very dense bone block will take 14 days or longer for complete decalcification .

The tissue must be suspended in a piece of gauze in the upper part of the jar in order that the dissolved salts sink in the bottom. The acid solution should be changed daily or even twice a day for better decalcification.

Acid decalcifying solutions:- 1.Nitric acid : use aqueous solution 5% nitric acid , change solutions every day or twice a day for 1-4 days. Disadvantage: Nitric acid produces a marked decrease in the stain ability of the tissue after 20 hrs. 2.Trichloro-acettic acid : use 10% solution. decalcification carried out in large quantities of the solution. 3.Formic acid : this is most suitable for decalcification and good preservation of stain ability even it has been necessary to leave bone in this acid for longer time .

Washing tissues: after tissues are decalcified , wash in running tab water for 3-8 hrs. to remove the last traces of the decalcification solution . Solutions may also be neutralized after decalcification with acid by immersing in a weak solution ( 2%) of lithium carbonate for a similar period of time. Staining The routine H&E stain gives satisfactory results after acid decalcification . Problems 1.Failure to stain properly ,most often due to over treatment in acid decalcifying solutions, or to insufficient washing out of the acid.( just before staining , the bone sections may be neutralized by placing the slides in a 1% aqueous solution of lithium carbonate). 2.Sections of bone are likely to float off the slide during staining , particularly following treatment in acid alcohol , the slides must be checked out after they have been deparaffinized , If bone sections show signs of loosening , it is best to celloidinize the sections before processing with the staining technique.

Determining the end point of decalcification ( make sure that no specimen is left in the decalcifying sol. overnight) 1.incerting a needle into the bone , if the needle enter the sections easily, the bone is ready to be processed . This method of testing be used 2.check by pliability of the tissue , if it bends easily and is flexible , it is decalcified. 3.chemically testing the solution to determine calcium loss . After a period of decalcification , 5 ml of decalcifying fluid is aspirated from the base of the jar containing the specimen . to this add 5 ml. of 5% ammonium oxalate and 5 ml. of 5% ammonium hydroxide , mix and allow to set for 10 – 30 minutes. If the solution is cloudy or turbid after the elapsed time , the decalcifying solution should be changed and the test made again after additional period of decalcification. The specimen is considered properly decalcified when clear solution is obtained.

Von Kossa Stain

This histology stain is a silver reduction method used to

visualize calcium and calcium deposits. Here the

chondrocytes in the growth plate are staining black.

CAEBOHYDRATES

Methods for Histochemical Demonesration of Carbohydrates

1- PAS reaction

Results: PAS +ve material = magenta

2- Best’s Carmine method :- is used for paraffin, frozen and freeze dried sections

Results: Glycogen will show the characteristic red stain. Nuclei stained blue.

3-Alcian blue –PAS method: Results

Acid Mucosubstances: blue

Neutral Mucosubstances: red

Mixture: purple

4- Acid Mucopolysaccharides: Toluidine blue

method

Results: Acid Mucosubstances pink nuclei blue.

5- Acid Mucosubstances: Alcian blue (pH 2.5 , pH

0.2)

Results: Alcian blue PH 0.2: sulphated acid

mucosubstances blue color

Alcian blue PH 2.5 most acid mucosubstances

appear blue.

6- Silver method for ascorbic acid(Bacchas 1950

and Jensen& Kavalijian 1956).

Results: ascorbic acid black.

NUCLEIC ACIDS

Methods for Histochemical

Demonstration of Nucleic Acids

1- Feulgen nuclear reation (DNA)

results: DNA appears reddish

purple.

When put in a light green stain as a

counterstain

cytoplasm: green

2- Methyl Green – Pyronin method (DNA

& RNA)

results: DNA : green RNA: red

The Methyl Green Pyronin (RNA DNA

Stain) is intended for use in the

histological visualization of

DNA, RNA and Mast Cell Granules.

3-Hibiscus method (DNA)

results: Nucleus: dark blue When put

in a eosin stain as a counterstain

cytoplasm: light red.

PROTEINS AND AMINO

ACIDS

1- Mercury-bromophynol blue method:-

Result: Proteins deep blue.