Introduction to histology, histological techniques

Outline

• Introduction
• Histological techniques
  • Biopsy and aspiration
  • Fixation
  • Dehydration
  • Infiltration
  • Embedding or casting
  • Sectioning and Mounting
  • Staining
  • Placing the slide on the microscope.
  • Cryofracture and freeze etching
  • Microwave method of tissue processing
  • Tissue culture
  • Autoradiography
  • Artifacts

Introduction

Histology is the study of the tissues of the body and how these tissues are arranged to constitute organs.

Tissues have two components , cells and cellular matrix (ECM)

The ECM consists of many kinds of macromolecules e.g. collagen fibrils. ECM is produced by the cells locally by an orderly combination of these tissues ,organs are formed.

ECM supports the cells and is the milieu for supply of nutrients and clearing of waste and secretory products. During development, cells and their associated matrix become functionally specialized and give rise to fundamental types of tissue with characteristic structural features.

Cells and tissues could be studied in vitro or in vivo.

In vivo histology includes USS, CT,RMI studies of tissues in the living being. In vitro histology consists of the method used to study tissues outside of the body

In vitro studies could be on living cells/tissue or dead cells/tissues

Histological Techniques

These are procedures used in the acquisition , processing and viewing of histologic samples If well carried out, yields quality results

Biopsy and aspiration

• Method of acquiring tissue sample for study.
• Small portion of specimen is excised with sharp blade or fluidy specimen aspirated with aid of needle
• Fresh tissue specimens could come from various sources.
• They can easily be damaged during removal from patient or experimental animal
• They should be handled carefully and fixed as soon as possible after dissection

Fixation

• Slide preparation begins with fixation of tissue specimen.
• To preserve tissue structure .
• Its purpose is to prevent tissue autolysis and putrefaction.
• For best results, biological tissue samples should be transferred into fixative immediately after collection.
• Although there are many types of fixative, most specimens are fixed in 10% neutral buffered formalin.
• The optimum formalin to-specimen volume ratio should be at least 10:1 (e.g., 10ml of formalin per 1cm3 of tissue).
• This will allow most tissues to become adequately fixed within 6-24 hours

Dehydration

• Water in a specimen must be removed before it can be infiltrated with wax.
• Carried out by immersing specimens in a series of ethanol (alcohol) solutions of increasing concentration.
• Ethanol is miscible with water and progressively replaces the water in the specimen.

Clearing

• Wax and ethanol are largely immiscible.
• Solvent miscible with both ethanol and paraffin wax is used to displace the ethanol in the tissue.
• Then this in turn will be displaced by molten paraffin wax.
• This stage in the process is called “clearing” and the reagent used is called a “clearing agent”.
• Clearing agents used include xylene and toluene

Wax infiltration

• The tissue can now be infiltrated with a suitable histological wax
• A typical wax is liquid at 60°C and can be infiltrated into tissue at this temperature.
• It is actually done in an oven

Embedding

• Process of enclosing tissue in the embedding mould.
• Mould is filled with melted wax and specimen is placed on it.
• Orient the specimen well on the mould.

Sectioning and mounting

• A microtome is used to slice thin tissue sections off the block in the form of a ribbon
• The microtome can be preset to cut different thickness

Staining

• Stains are dyes applied to tissue specimen to make them conspicuous and distinguishable from one another.
• Anionic cell components such as nucleic acid have affinity for basic dyes hence called basophilic.
• Cationic cell components , such as proteins stain with acidic dyes and are termed acidophilic
• Basic dyes include , hematoxylin,tolidine blue ,alcian and methylene blue
• Acidic dyes include eosin,orange G and acid fuchsin
• A combination of stains further distinguishes more tissue components eg H/E
• In a combination ,the second stain is called a counterstain

Staining

• Most cells are transparent, and appear almost colourless when unstained.
• Haematoxylin and Eosin are used to provide contrast to tissue sections
• Making tissue structures more visible and easier to evaluate.
• Following staining, a cover slip is mounted over the tissue specimen on the slide
• Optical grade glue is used to help protect the specimen.

Placing the slide on the microscope

• After staining, slide is allowed to dry.
• Afterwards the slide is ready for viewing on the microscope.
• The stained slide is placed on the stage of the light microscope and adjusted accurately for viewing.

Cryofracture and freeze etching

• This a technique that allow microscopy of cells without fixation or embedding
• Useful in the study of membrane structure
• Very small tissue specimen is rapidly frozen in liquid nitrogen then cut or fractured
• The small slice could be viewed under microscope

Microwave method of the tissue processing

• This is a recent technique, in which the microwaves possess penetrative properties with shorter processing time (Hegazy et. al., 2015)
• The size of tissues used must not be more than one cubic cm; otherwise complete and even penetration of microwaves will not occur.
• Has the advantage of reducing processing time
• May alter heat labile structures

Cells and tissue culture

• Live cells and tissues are maintained and studied outside the body in culture.
• Cell culture allows the direct observation of cellular behavior under the microscope.
• Sample of culture is mounted on the slide for viewing

Autoradiography

• Autoradiography is a technique that uses photographic film to determine where within a cell a specific radioactively labeled compound is at the time the cell is fixed and sectioned for microscopy

Enzyme histochemistry

• This is a method for localizing cellular structures using specific enzymatic activity
• Examples of enzymes detectable are phosphatases , dehydrogenases and peroxidase

Artifacts

• Artifacts are the result of changes in a tissues structure or the addition of new structure. They include:
• Swelling and Shrinkage of tissue components a result of poor fixation and/or dehydration techniques.
• Swelling and shrinkage can sometimes result in rupture of membranes. This sort of damage is particularly evident at the ultrastructure level.
• Wrinkles in section, Tear in section, Air bubbles and Dust: Usually the result of poor sectioning technique or poor technique during mounting of sections.
• Stain precipitate: Can result from use of old stain solutions, use of unfiltered stain solutions.