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Tissue Preparation For Light Micros

Summary of tissue processing & H&E staining for light microscopy, suitable for students of medical & allied fields.

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4K views

Tissue Preparation For Light Micros

Summary of tissue processing & H&E staining for light microscopy, suitable for students of medical & allied fields.

Uploaded by

scribdarnab
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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TISSUE PREPARATION FOR LIGHT MICROSCOPY

TISSUE PREPARATION FOR LIGHT MICROSCOPY


Tissues are made of two interacting components: cells, & extracellular matrix.
Slides used in the histology lab are of formalin-fixed, paraffin-embedded, hematoxylin & eosin
(H&E)-stained tissues.
Steps in preparation of tissues for examination by a (bright-field) compound microscope, &
reagents used in each step, are:
Fixation (formalin)
Dehydration (ethanol)
Clearing (xylene)
Infiltration (paraffin)
Embedding (paraffin)
Sectioning (rotary microtome)
Staining (H&E)
Mounting (resin)

FIXATION

To preserve tissue structure. (Ideally the microscopic tissue preparation will have the same
structure & molecular composition as it had in the body). Fixation:
Stops cell metabolism
Prevents autolysis
Kills pathogenic microorganisms
Hardens tissue (by cross-linking proteins)
Increases the tissues receptiveness to further processing.
The most commonly used fixative is 10% neutral buffered form (NBF phosphate buffer)
formalin. Formalin is a 37-40% (saturated) aqueous solution of formaldehyde gas. Therefore
10% formalin means 4% aqueous formaldehyde solution.
Formalin preserves the structure of cells & extracellular components by reacting with amino
groups of proteins, cross-linking them:
Intracellular cytoskeletal proteins
Extracellular structural proteins like collagen
Nucleoproteins
Membrane proteins
Dr A Bhar

TISSUE PREPARATION FOR LIGHT MICROSCOPY


Formalin does not react with lipids & carbohydrates. Lipid components such as membranes &
carbohydrates like glycogen, mucinogen, & proteoglycans are not well preserved with formalin
fixation. To retain these components other fixatives need to be used. Osmium tetroxide, used
as a fixative in electron microscopy, preserves lipids. Another method is to use frozen sections
(which need to be sectioned with a cryostat).

DEHYDRATION

Formalin is an aqueous solution. The embedding medium paraffin is not soluble in water.
Therefore water has to be removed from the tissue. This is done by immersion in graded
(ascending) concentrations of a dehydrating agent, most commonly ethanol, at 70%, 90%, &
100% concentrations. Direct use of 100% ethanol will cause cell damage. For delicate tissues,
dehydration is started at a 30% or 50% dilution. Water in the tissue is replaced by ethanol.

CLEARING

Clearing agents include xylene & toluene, which act as an intermediary between the
dehydrating agent & the embedding agent, and are miscible with both. Immersion in xylene
replaces the ethanol with xylene, which is replaced by paraffin in the embedding stage.

INFILTRATION & EMBEDDING

The cleared tissue is placed in a mold and liquid paraffin (a mixture of long-chain hydrocarbons
produced in the cracking of mineral oil; M.P. variable ~ 47-64C) added. Liquid paraffin
infiltrates tissues & replaces xylene, solidifying when cooled. This prevents distortion of tissue
structure during microtomy. During embedding the tissue has to be placed in the proper
orientation in the mold.

SECTIONING

Thin sections (5-15m) are cut using a microtome. Different types of microtome are available.
A rotary microtome is usually used for paraffin sections.

The paraffin block is taken from the mold & attached to a chuck that can be placed on
the microtome.
The block is trimmed to appropriate size, placed on the microtome, & a ribbon of
sections (of 3-4m thickness) are cut.
The free end of the ribbon is held by fine pointed forceps or a teasing needle, while
the other end is detached from the microtome knife with a sable or camel hair brush.
The ribbon is placed in a tissue-floatation bath carefully, to smooth out any wrinkles in
the ribbon. The temperature of the bath should be 10C less than the melting point of
the paraffin. [A drop of alcohol or detergent can be added to the bath to reduce the
surface tension, allowing the ribbon to flatten out with greater ease].
After 30 seconds, sections are picked up onto clean albuminized (adhesive) slides.
Universal size of standard slides is 76x25mm, with a thickness of 1.0-1.2mm. The
slides are labelled with chemical-resistant pen or pencil, & placed on a hot plate or in

Dr A Bhar

TISSUE PREPARATION FOR LIGHT MICROSCOPY


a drying oven, whose temperature is set at the melting point of paraffin. Overnight
drying at 37C is used for many tissues. If the temperature is too hot, distortion of cells
will occur.

STAINING

CHEMICAL BASIS OF STAINING

Three questions need to be answered:

1. Why do any tissue components stain?


2. Why do stained components remain stained?
3. Why are all components not stained?

Tissue components stain because of:

1. Stain-tissue interactions. Four types of chemical interactions may occur ionic, van
der Waals, hydrogen bonding, & covalent bonding.
2. Solvent-solvent interactions. Hydrophobic effect (e.g. between adipose tissue & Sudan
dyes).
3. Stain-stain interactions. Dye molecules tend to form aggregates. This effect is more
prominent in aqueous solution, where the hydrophobic effect is present. Van der Waals
interactions are also important, in both aqueous & non-aqueous environments. With
basic (cationic) dyes like toluidine blue, this occurs on substrates of high negative
charge, like sulfated proteoglycans in mast cell granules, hyaline cartilage matrix.
This causes metachromatic staining (purple instead of blue), because the dye
aggregates have spectral properties different from the monomeric dye.

The most commonly used staining method uses hematoxylin & eosin (H&E) stains.
Dr A Bhar

TISSUE PREPARATION FOR LIGHT MICROSCOPY


Hematoxylin (extracted from the wood of a tree) itself is not a stain. Its oxidation product
hematin, is an anionic (basic) dye that will stain acidic cellular components (like DNA, RNA)
purple. It is used to stain the nucleus. A mordant (alum is commonly used) is required to
confer a net positive charge to the dye-mordant complex so that it can bind to anionic tissue
sites. Hematoxylin is soluble in water. Hematoxylin is a regressive stain, meaning all tissue
components stain, but during washing stain is removed from components with which it
interacts weakly (as opposed to a progressive stain, where intensity of staining increases
progressively with exposure time).

Eosins are (acidic) xanthene dyes that stain (basic) cytoplasmic & extracellular connective
tissue components (matrix & fibres) pink. Eosin Y (yellow) is used commonly, which is soluble
in water, & also in alcohol. Eosin is used as a counterstain after staining with hematoxylin.

Basophilic components of the cell & extracellular matrix (that stain purple with hematoxylin)
include:

Nuclear components: heterochromatin & nucleoli


Cytoplasmic components: RER (because of RNA)
Extracellular materials: cartilage matrix

Acidophilic components of the cell & extracellular matrix (that stain pink with eosin) include:

Cytoplasmic components: cytoplasmic filaments (e.g. actin & myosin in muscle cells)
Extracellular materials: most extracellular fibres (e.g. collagen) because of amino
groups.

PROCEDURE

Before staining the paraffin has to be dissolved out of the tissue using xylene or toluene.

Hematoxylin is soluble in water. Xylene has to be removed & the tissue rehydrated using
graded dilutions of alcohol (100%, 90%, 70%) before staining with aqueous hematoxylin. After
hematoxylin staining the slide is washed & counterstained with aqueous eosin.

MOUNTING

A drop of mounting medium (e.g. DPX resin) is placed over the tissue & covered with a cover
slip. If the mounting medium is resin-based (non-aqueous), then the slide has to be dehydrated
again using graded concentrations of alcohol, then alcohol removed with xylene, before the
mounting medium can be applied.

Dr A Bhar

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