METHODS USED IN HISTOLOGY

TISSUE PREPARATION,
HISTOCHEMISTRY, CYTOCHEMISTRY
 Main Groups

1. Methods for the observation of living cells

2. Methods for the observation of dead cells

 Living Cells

• Cells from tissue cultures and unicellular organisms

can be placed on a glass slide and observed with a
phase-contrast microscope or confocal microscope

•Tissues such as the mesentery and others can be used

 Vital and Supravital Stains
Vital stains are harmless substances injected in intact animals and are taken by specific
tissues
• Trypan blue

Unable to pass thru the plasma membrane

Live cells stained negatively

Supravital stains dye live cells previously removed from the organism
more toxic than vital stain few cells survive the stain
• Methylene blue
 Sources of Samples

•In medical practice, the main sources of samples are:

•Biopsies – surgical specimens

•Autopsies – post-mortem specimens

•Cytology – blood or other sources [Papanicolaou (PAP) smears]

•Karyotyping-chromosome analysis-ploidy
Basic Steps UsedLight
in Tissue Preparation
microscopy

● Sampling
● Fixation
● Dehydration
● Clearing
● Infiltration (impregnation)
● Sectioning
● Staining
● Mounting
Basic Steps Used in Tissue
Preparation
 Sampling

•Aim – to provide a representative specimen

•If possible, the sample should come from a living organism
•If not possible, then after a short post-mortem interval
•Be careful during the removal
•Choose a representative portion of the sample
 Precaution !
•Always handle tissues with the proper safety measures, since they might be
INFECTED.
 Fixation
Aim – to preserve the cells with the least alteration possible
from the living state
– Use of substances that coagulate the protoplasm
1. Simple – formaldehyde, alcohol, acetic acid, osmic acid,
picric acid
•10 % formalin is the cheapest and easiest to keep fixative solution, although it does
not fix / react with lipids.

2. Complex – Bouin’s fluid, Zenker’s fluid, special mixtures

(should be buffered)
 Dehydrating

Aim – to take the water out of the sample to allow paraffin to

impregnate the tissue.
The sample goes through increasing concentrations of alcohol
Usually, dehydration starts with 60 % ethanol, and the sample goes
through several changes, until it reaches the 100 % ethanol
concentration
 Dehydrating
•The smaller the increase of alcohol concentrations, the lesser the chance of shrinkage of the tissue.

•Lipids are eliminated with alcohols

 Clearing

•Aim – to take the alcohol out of the sample to allow paraffin to impregnate
the tissue

•The tissue is sunken in xylene (xylol) that is miscible both in the

dehydrating and the embedding agent.

•Benzene, cedarwood oil, chloroform etc.. can also be used

 Infiltration

•Aim – to embed the sample in a material that allows easy

cutting
•The sample goes inside a bath of warm paraffin
•The tissue embedded in paraffin is put in a plastic frame that can
be used later in the microtome
 Sectioning
Aim – to cut the sample in slices thin enough to be useful

Sections of the tissue are cut with the aid of the microtome
The thickness, between 3 and 10 µm, is selected depending on
the stain to be used
The section is transferred to a glass slide using albumin as
adhesive
Microtome
Staining
•After the sections are put on the glass slide, the process shall be reversed in order to remove
the paraffin and hydrate the tissue again
 Staining cont.
Aim – to add color to the structures of the sample in order to
differentiate them
There are several phases of staining: take out the paraffin,
rehydrate, stain with hematoxylin, then with eosin

Every stain has a different procedure (phases, time, extra

procedures, etc.)
Most common stain is hematoxylin and eosin (H&E)
Modern slide stainers
 STEPS IN STAINING PARAFFIN SECTIONS

1. SECTION ON THE GLASS SLIDE

2. REMOVING OF WAX WITH XYLENE

3. REMOVING OF XYLENE WITH ABSOLUTE

ALCOHOL

4. HYDRATION IN DESCENDING GRADES OF

ETHANOL
1. STAINING

2. DEHYDRATION IN ASCENDING GRADES

3. CLEARING IN XYLENE

4. MOUNTING
 Mounting
Aim – to put the final sample in a medium that
can protect it and also be used in the light
microscope

•A drop of mounting agent with a similar

refractive index to that of glass is placed on the
stained section, and then a glass coverslip is
added

•Canada Balsam in xylene is commonly used

mounting media. Permount is also used.
H & E staining
HAEMATOXYLIN & EOSIN
◦ MOST COMMONLY USED STAIN - LM

◦ HAEMATOXYLIN
◦ BASIC DYE
◦ STAINS ACIDIC PART OF CELL
◦ STAINS NUCLEUS OF CELL PURPLE/ BLUE

◦ EOSIN
◦ ACIDIC DYE
◦ STAINS BASIC PARTS OF CELL
◦ STAINS CYTOPLASM PINK
H&E
 Freezing Methods
Aim – to quickly prepare a glass slide for rapid
study of specimens
- may be used during a surgical procedure

• The tissue is first frozen, then sectioned

while frozen (frozen section)
• Section is placed on glass slide and stained.
• Examine with microscope
• The quality of the specimen may be better with
routine formalin processing
Cryotome
 Artifacts
• Improper histological techniques are the usual cause

➢ The main cause of artifacts is poor fixation

➢ Autolysis – poor preservation of tissue
➢ Poor sampling – not putting in proper fixative
➢ Shrinkage – dutin prep – conc of solutions
➢ Folds – when put in water bath
➢ Stain precipitation and dust / debris
➢ Defects in the knife
➢ Detachment of layers
Suprarenal gland with
autolysis
villi

mucosa

muscularis
Folds
Autolysis and tissue fragmentation
 Special Techniques
1. Histochemistry
2. Enzyme digestion
3. Enzyme histochemistry
4. Immunohistochemistry –
5. Autoradiography

Most of this techniques are only used in research

 Histochemistry
Different components of tissues and cells reacts
with some chemicals
This property is used to stain those components
For example, in the Perls’ reaction, ferric ions
react with potassium ferrocyanide to create
insoluble ferric ferrocyanide
 Enzyme Digestion

•The use of an enzyme to assess if the

substance stained is really what we are
looking for.

•For example, if one stains for DNA and after

pretreatment with DNAse, one cannot stain
anymore, then the staining is for DNA.
 Enzyme Histochemistry

The enzymatic activity will help to stain the

structure or the cellular region you want to
analyze.
ATPase STAIN OF MUSCLE DEMONSTRATES TWO
TYPES OF FIBERS, FAST AND SLOW TWITCH
chromatin
 Immunocytochemistry
immunohistochemistry
•An antibody conjugated with a fluorescent dye is
applied to a sample

•Some substances of the sample act as antigens,

therefore fixing the antibody

•A UV microscope is used to make the dye apparent

 Autoradiography

Molecules are marked with tracer

radioactive isotopes
They get incorporated into cellular
structures
The slide is dipped in
photographic emulsions
The silver grains in emulsion
appear as dark grains. Autoradiography of formalin-fixed mouse vaginal
epithelium shows the location of [3H]thymidine-
labeled DNA
Micro-villi
 Historadiography

Used to study bone and other calcified tissues

Mostly used in research of mineral density