Micro-Techniques Presented By :

Dr.Tareq Abu-Libdeh

❖ Paraffin technique

✓ Technique used to prepare the tissues for light microscopy

( tissue can be obtained from 1-cadavir 2- biopsy 3- experimental animal)

▪ it includes the following steps:

1. Fixation : in appropriate solution (formol saline)
2. Dehydration and clearing : in alcohol then xylol
3. impregnation & Embedding : in paraffin wax (Soft & hard)
4. Sectioning : by microtome
5. Mounting : on glass slides
6. Staining of the sections
 Micro-Techniques Presented By :
Dr.Tareq Abu-Libdeh

1- Fixation

• To maintain the structure of the tissue as in the life state

• Immediately the sample for analysis is placed in a fixative solution

upon removal from the body

• Fixation is done as soon as possible to prevent autolysis and to preserve the morphology.

For LM:

✓ Formol saline

For EM: a mixture of

✓ Glutaraldehyde

✓ Osmium tetroxide

Advantage of fixation :
✓ Hardens the tissue by coagulating its protein
→ Facilitate the process of cutting & staining & examination

✓ Prevent putrefaction & stop autolytic changes by killing bacteria

✓ Preserves the molecular & morphological structure of the tissue

( but not the chemical)
 Micro-Techniques Presented By :
Dr.Tareq Abu-Libdeh

2- Dehydration & Clearing

Dehydration : Is done by treating the specimen with ascending concentration of alcohol
( 50% → 70%→ 100%)
Gradual removal of water from the specimen ( H2O represent 70% of the body)

Clearing : with this process the tissue become translucent

(to allow the light to penetrate the specimen)
the tissue is treated with xylol or benzol … to remove the alcohol

3- Impregnation & Embedding

Impregnation:
✓ Tissues are placed in molten soft paraffin wax
✓ The wax infiltrates the tissue
& occupies all the spaces that were originally occupied with water
Embedding:
✓ Tissue are placed in molten hard paraffin wax
✓ The tissue is placed in the center of the paraffin,
which hardens as it cools → paraffin block

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 Micro-Techniques Presented By :
Dr.Tareq Abu-Libdeh

Automatic tissue processor

The steps required
to take animal or human tissue
from fixation
to the state where it is
completely infiltrated with soft paraffin wax
then to be embedded in hard wax
for section cutting on the microtome.

4- Sectioning by Microtome

❖ A microtome is a mechanical device

used to cut extremely thin slices of a fixed tissue block known as sections.

❖ It holds the block of hard paraffin with the tissue in its center
against a sharp metal knife that used to cut the block into thin sections (3-10 microns)
as it moves up and down

Prof.Dr. Hala 1
 Micro-Techniques Presented By :
Dr.Tareq Abu-Libdeh
5- Mounting
• Tissue sections are placed on glass slides smeared with egg albumin
• then warmed on a hot plate to dry.
• The sections are now ready to be stained

Note:
These steps take around 2-3 days (disadvantage)

❖ Freezing technique

• Fresh frozen tissues are cut using freezing microtome (cryostat).

• The sections placed in cold fluid called Isopentane/liquid nitrogen (-50 C)
• then rapidly stained.

Advantages:
✓ Rapid technique for diagnosis of tumors. ( Intraoperative )
✓ No fixation , No dehydration & No chemicals are used,
✓ so useful for histochemical (enzyme staining) studies.

Disadvantages:
✓ Non serial & Fragmented sections .
✓ Cannot be preserved for a long time.
 Micro-Techniques Presented By :
Dr.Tareq Abu-Libdeh
6- Staining

▪ The tissue sections that will be studied using the light microscope
must be stained first since most tissues are colorless
▪ Dyes (stains) used are either basic or acidic
 Micro-Techniques Presented By :
Dr.Tareq Abu-Libdeh
❖ Common (Routine) histological Stains – H&E

• Hematoxylin (H) : • Eosin (E):

▪ blue basic dye ( +ve charged) Red acidic dye (- ve charged)
▪ Stains acidic (anionic –ve) Stains basic (cationic +ve)
components of the cell with a components of the cell with
ared color
blue color
e.g. cytoplasm,
▪ e.g. nucleus, ribosomes (r-RNA) mitochondria, muscles !!
▪ Basophilic structure = blue (it has +ve charged proteins)
Acidophilic structure=red

L.S skeletal ms. fiber ( contains actin & Liver cells contains plenty of
myosin) stained with H &E mitochondria stained with H &E
 Micro-Techniques Presented By :
Dr.Tareq Abu-Libdeh

Collagen fibers stained with H & E

Automatic slides staining machine

❖The clinical values of special stains

▪ Special stains answer specific questions like what type of cells and tissues
▪ Used in the diagnosis of medical diseases
like Trichrome stain in case of Liver Cirrhosis
 Micro-Techniques Presented By :
Dr.Tareq Abu-Libdeh
Vital stain:
▪ Stain the living cells inside the living animal.
▪ Done by injecting the dye into living animal prior to examine the tissue
▪ .e.g. staining phagocytic cells (macrophages) with Trypan blue& Indian ink

Leishman stain:
▪ mixture of acidic & basis stains
to stain nuclei & cytoplasm
(methylene blue & Eosin) It used to stainblood
films to demonstrate
white blood cells, malaria parasite

Note : the RBCs are anucleated cells , so they won't take the blue stain

Red blood cells

Technique of blood film infected with malaria parasite

Metachromatic stain:
▪ Stain gives the tissue new color different from that of the original stain
e.g. Toluidine blue when stains Mast cells gives purple color
(different from the blue color of the stain). Phenomenon called metachromasia
 Micro-Techniques Presented By :
Dr.Tareq Abu-Libdeh
Trichrome stains: (connective tissue):
▪ 3 stains used in combination to stain different tissues Components
▪ e.g. collagen fibers stained blue

Orcein stain:
stains elastic fibers brown ( wall of aorta)

Silver (Ag) stain:

nerve cell brown & reticular fibers black
 Micro-Techniques Presented By :
Dr.Tareq Abu-Libdeh
Histochemical stains: (Relate structure to function)
technique used selectively identify & demonstrate the distribution of chemical
compounds or enzymes within and between the cells
e.g. mucine or alkaline phosphatase enzyme
Concept: enzyme of interest in a cell or tissue converts its substrate to a
colored or florescent product that can be visualized at the site of the activity

Histochemistry (Alkaline phosphatase enzyme)

this enzyme removes phosphate group from protein

Immuno-histochemical (IHC) stains: cryostat

 Laboratory method that selectively identify antigens
 using specific antibodies to check for these antigens in a sample of tissue.
 The antibodies are usually linked to an enzyme or a florescence dye (markers)
(Labeled antibodies).
 After the antibodies are linked to the antigen in the tissue sample , the enzyme or
dye is activated. The localization of the antigen can be seen under the microscope
 IHC staining can be detected through 2 methods of detection either
chromogenic and florescent (labeling we use : Fluorescein , Rhodamine)
 Indirect vs direct IHC : high sensitivity , signal amplification,
ability to detect several targets in the same sample
 This is method use for:
✓ Diagnosis of cancers ( markers)
✓ and can tell the difference between different types of cancer .
✓ Tumor specific

Florescein
Microscope

Immunohistochemistry
 Micro-Techniques Presented By :
Dr.Tareq Abu-Libdeh

Molecular analysis
✓ It means biochemical analysis of certain components of the cell.
✓ It is usually quantitative in nature.

✓ Examples are:
✓ Protein-electrophoresis
✓ DNA – electrophoresis
✓ Fluorescent In situ hybridization ( FISH technique)
✓ Detection of certain ions in the cell e.g. Ca, Fe….etc.

Protein electrophoresis: (BL serum protein electrophoresis)

▪ Proteins carry a positive or a negative electrical charge,

and they move in fluid when placed in an electrical field.
▪ Proteins will be separated according to their charge & molecular weight
▪ ( e.g. blood cancers:↑ increased gamma globulin protein (M protein)
= multiple myloma)
 Micro-Techniques Presented By :
Dr.Tareq Abu-Libdeh

DNA Ladder: DNA fragments of

DNA electrophoresis: known lengths used to estimate
the size of unknown DNA molecule
▪ is technique used to identify & quantify DNA fragments
(DNA fragments are -ve charged)

▪ (in this case separation is based on length of the base pair)

▪ Samples are loaded into wells of an agarose or acrylamide gel

and subjected to an electric field,
causing the negatively charged nucleic acids
to move toward the positive electrode.
▪ Small fragments will move faster than the large ones

▪ ( DNA fingerprint , gene isolation, disputed paternity)

Fragment size usually

referred to as base pair (bp).
The shorter fragments
travel faster
 Micro-Techniques Presented By :
Dr.Tareq Abu-Libdeh

Fluorescent In situ hybridization ( FISH technique):

▪ Molecular technique used to visualize and map the genetic material.
▪ Use to localize the site of the genes on chromosomes
using a fluorescent probe

fluorescent probe:
▪ fragment of DNA or RNA of variable length
which can be radioactively labeled (probe)

▪ It can be used in DNA or RNA samples

to detect the presence or absence of nucleotide

▪ sequence that are complementary

to the sequence on the probe .

▪ Useful in detect chromosomal abnormalities

Florescein
Microscope
 Micro-Techniques Presented By :
Dr.Tareq Abu-Libdeh

Methods for study tissues

 In vivo studies: within the living body .

Stuty of tissues after doing any experiment
inside the living body
(animal model based testing)

 In vitro studies: outside the body

Study of tissues outside
their normal biological context ( cell based testing )

Cell and Tissue Culture

✓ In vitro cultivation of tissues & cells at defined temperature (37C)

using an incubator & supplemented with a medium containing cell nutrients & growth
factors(like animal serum) is collectively known as tissue culture.

✓ Different types of cells can grow in cultures as: white blood cells, fibroblasts, skeletal and
cardiac muscle, epithelial tissue (liver, breast, skin, kidney)
and many different types of tumor cells.

❖ Medical uses of tissue culture:

1. used in studying chromosomal patterns of individuals ….Karyotyping, gene therapy

2. Used in researches of cancer

3. Used in cultivation of bacteria, viruses, in order to prepare different vaccination

4. Study the effects of new drugs

 Micro-Techniques Presented By :
Dr.Tareq Abu-Libdeh
Cell culture
 Cells can be isolated from the body for in vitro cultures.
 Cells can be released from tissues by enzymatic digestion
 Using enzymes such as collagenase and trypsin which break down the extracellular matrix.

Primary cultures
Refer to the cells that are cultured
directly from a tissue (parent cells)

Secondary cultures
Once the parent cells reach confluence they have to be sub- cultured
(i.e. passaged) by transferring them to a new vessel with fresh growth medium to
provide more room for continued growth

Confluence
stage in which the cells (1ry or 2ry) become adherent to & covering most of the
culture surface forming monolayer ( e.g. 25%,50%, 100%)

Cell passage = subculture Different degrees of confluency

 Micro-Techniques Presented By :
Dr.Tareq Abu-Libdeh

cell line
✓ a population of cells (clone) developed from a single cell
therefore consisting of cells with a uniform genetic make-up
(phenotype & function)
✓ Cell lines have a limited life span, and as they are passaged

immortalized cell line

✓ Has acquired the ability to proliferate indefinitely.
✓ It is obtained from subcultures of the primary culture
✓ Normal immortalized cell line: stem cells
✓ Abnormal immortalized cell lines : cancer cells

Cell fractionation
✓ It means isolation of the cell components (nucleus & organelles)
while preserving its individual function to study the features of each.

✓ This is done by the use of centrifugation at different speeds and periods of time. The
factor that determine whether a specific cell component ends up in the supernatant or
the pellet is size and weight of component

✓ Nuclei are the first to be separated followed by different cell organelles

✓ The true arrangement in centrifugation:

Nuclei ––> Mitochondria ––> Microsomes ––> Ribosomes

The sediment at the bottom of the tube is called pellet,

the less dense component at the top is called supernatant