Introduction to Histological Techniques

Teaching Team

  • Dr Lie Chen (lie.chen@ubd.edu.bn)

  • Dr Faizah Naim (faizah.naim@ubd.edu.bn)

  • Dr Shirley Lee (shirley.lee@ubd.edu.bn)

  • Guest lecturer: Dr. Steven Tan (RIPASH)

Module Timetable

  • Fresher's Week (02-01-2024 to 07-01-2024)

  • Week 1:

    • Date: 14-01-25

    • Time: 9-11 am

    • Session: Lecture

    • Topic: Introduction to “Cellular Pathology “ Histological Techniques

    • Lecturers: Dr Lie, Dr Shirley

  • Week 2:

    • Date: 21-01-25

    • Time: 9-11 am

    • Session: Lecture

    • Topic: Methodological Development in Histopathology

    • Lecturer: Dr Faizah

  • Week 3:

    • Date: 28-01-25

    • Time: 9 – 11.50 am

    • Session: Practical

    • Topic: Staining of Haematoxylin & Eosin (H & E), Periodic Acid Schiff (PAS) and Alcian Blue

    • Lecturers: Dr Faizah, Dr Shirley

  • Week 4:

    • Date: 04-02-25

    • Time: 9-11 am

    • Session: Lecture

    • Topic: Epithelial Tissues

    • Lecturer: Dr Lie Chen

  • Week 5:

    • Date: 11-02-25

    • Time: 9-11 am

    • Session: Lecture

    • Topic: Connective Tissue

    • Lecturer: Dr Shirley

  • Week 6:

    • Date: 18-02-25

    • Time: 9-11 am

    • Session: Lecture

    • Topic: Muscle and Nerve

    • Lecturer: Dr Lie Chen

  • Week 7:

    • Date: 25-02-25

    • Time: 9 – 11.50 am

    • Session: Practical

    • Topic: Observation of Epithelium, Connective Tissue, Bone and Muscle

    • Lecturers: Dr Lie Chen, Dr Shirley

  • Mid Semester Break (03-02-2025 to 09-03-2025)

  • 4 MC module:

    • 9 Lectures

    • 4 Lab Practicals

    • 1 Case Study

    • Lecture Venue: Seminar Room D, L4- 5, Level 4 Extension Building

    • Practical Venue: L4-7, IHS Extension Building

  • Week 8:

    • Date: 11-03-25

    • Time: 9-11 am

    • Session: Lecture

    • Topic: Clinical Application of Cytopathology

    • Lecturer: Dr Steven

  • Week 9:

    • Date: 18-03-25*

    • Time: 9 – 11.50 am

    • Session: Practical

    • Topic: PAP Staining of Buccal Smear

    • Lecturers: Dr Faizah, Dr Shirley

  • Week 10:

    • Date: 25-03-25

    • Time: 9-11 am

    • Session: Lecture

    • Topic: Cellular adaptation and injury

    • Lecturer: Dr Lie Chen

  • Week 11:

    • Date: 01-03-25*

    • Time: 9-11 am

    • Session: Lecture

    • Topic: Carcinogenesis

    • Lecturer: Dr Lie Chen

  • Week 12:

    • Date: 08-04-25

    • Time: 9 – 11.50 am

    • Session: Lecture

    • Topic: Inflammation and Repair

    • Lecturer: Dr Faizah

  • Week 13:

    • Date: 15-04-25

    • Time: 9-11 am

    • Session: Practical

    • Topic: Observation of Squamous Cell Carcinoma & Adenocarcinoma

    • Lecturers: Dr Lie Chen, Dr Faizah

  • Week 14:

    • Date: 22-04-25

    • Time: 9-11 am

    • Session: Case study

    • Topic: Revision & Feedback Session

    • Lecturer: Dr Lie Chen

  • Revision Week (28-04-2025 to 04-05-2025)

    • Due to public holiday, the practical session will be rescheduled (date/time TBC).

Summative Examination

  • 60% [1 Physical/Written Exam]

    • Examination: May 2025 (Exact date TBC)

  • 40% [4 Practical Assignments]

Learning Outcomes

  • Introduction to cellular pathology

  • Steps in histological tissue preparation

  • Types of fixatives

  • Factors that affect optimal fixation of tissue

  • Basic principles of bright field microscopy

  • Microscopic techniques

  • Key stains used in histology, application, and principles

Pathology Definition

  • The scientific study of disease.

  • The medical characteristics of a disease.

Cellular Pathology

  • Study of disease in organs, tissues, and cells.

  • Histopathology and cytopathology are key diagnostic tests in the initial detection and diagnosis of cancer to inform therapy and patient management.

  • Also includes forensic pathology, neuropathology, pre-natal, perinatal and paediatric pathology, and oral pathology

Two Main Branches of Cellular Pathology

  • Cellular Pathology

    • Histopathology (study of tissue sections which contain many cell types)

    • Cytopathology (study of individual cells or cell cluster)

What is Histology?

  • Histology: microscopic study of structure of cells, tissues and organs in relation to their function

  • Human eye-macroscopic study (limited to objects with diameter > 200 μm)

  • Histology: Prepare thin specimens/sections (allows light to penetrate) of tissues (5-10 μm thick) for microscopic examination

  • Identify unknown specimens (normal vs pathogenic specimens)

  • Disease diagnosis (histological type of cancer)

Size Examples

  • Diameter of Single Human Hair: 40-20 μm

  • Human Red Blood Cell: 5-10 μm

  • Spider Web Silk Thread: 2-3 μm

  • Bacteria: 1 μm

  • Virus: 30-50 nm

Histology vs Histopathology

  • Histology is the study of tissues and their structure (correlate structure to function of tissues).

  • Histopathology is the study of tissues affected by disease.

Histological Grade of Breast Cancer

  • Histological grade of breast cancer as assessed by the Nottingham Grading System.

    • (a) A well-differentiated tumor (grade 1) that demonstrates high homology to the normal breast terminal duct lobular unit, tubule formation (>75%), a mild degree of nuclear pleomorphism, and low mitotic count.

    • (b) A moderately differentiated tumor (grade 2).

    • (c) A poorly differentiated (grade 3) tumor with a marked degree of cellular pleomorphism and frequent mitoses and no tubule formation (<10%).

Hierarchy of Anatomical Organization

  • Cell - fundamental unit of the organism; over 300 cell types in humans.

    • e.g., goblet cells - epithelial glandular cells of digestive and respiratory tracts. Goblet cells produce mucous secretion that provide lubrication of lumenal surfaces

  • Tissue - group of cells with similar structure and function

    • e.g., epithelial lining of the gut tract. Epithelium forms selective barrier to lumenal contents

  • Organ - group of tissues that collectively perform a common function

    • e.g., small intestine consists of collections of epithelia, connective tissue, muscle and nervous tissue. Small intestine is the site of enzymatic digestion and absorption

  • Organ system - series of functionally interrelated organs e.g., digestive system breaks down food items into simpler molecules

Classification of Tissues

  • Tissue: a group of cells and surrounding extracellular matrix (ECM)

  • Four primary types of tissue:

    • Epithelial tissue

    • Connective tissue

    • Muscle tissue

    • Nervous tissue

The Extracellular Matrix (ECM)

  • A 3-D network of non-cellular extracellular macromolecules, such as collagen, enzymes, and glycoproteins

  • ECM provides structural and biochemical support to surrounding cells by anchoring cells

Role of a Histology Student

  • Remember 3D characteristics of organ (shape, other organs/structures in close proximity)

  • Relate tissue structure and function

  • Observe specific cellular structures enhanced by particular stains (most common (routine) stain is H & E : Haematoxylin and Eosin) and connect the dots….

Distinguishing Different Tissue Components

  • Tissue components: hard to distinguish using a basic light microscope (little colour and contrast)

  • Various types of microscopes and methods of preparing/staining specimens for examination developed

Disease Diagnos tissues from body -> Process tissue in the lab (Fixation -> Dehydration, Clearing, Infiltration & Embedding -> Sectioning & Staining) -> Microscopic examination of section/specimen -> Diagnosis of disease.

  • After sectioning, tissue sections in wax are mounted to microscope slides before staining

Tissue Processing

  • Fixation

  • Dehydration

    • Water molecule is removed from tissue

  • Clearing

    • Dehydrating agent is replaced by clearing agent

  • Infiltration

    • Tissue is infiltrated with a supporting medium

  • Embedding

Anatomic Pathology Tissue Specimen Workflow

  1. Accessioning

  2. Grossing

  3. Tissue processing

  4. Embedding

  5. Sectioning

  6. Staining

    • Special Stains

    • Immunohistochemistry

    • In situ hybridization

  7. Reporting

Accessioning

  • Important that a tissue is identified and labelled correctly before being processed for diagnostic purpose

    • Identification:

      • i) Patient information

      • ii) Patient’s medical history

      • iii) Description of site of origin (of the tissue)

    • Labelling: Assign each specimen a number/identifier code

Grossing of Specimen

  • “Grossing" means inspecting the specimens, describing and measuring the tissue, inking if needed, and sectioning (trimming) the tissue to be processed for diagnosis.

  • Tissues selected for processing will be placed on cassettes (blue perforated baskets on the right image) and loaded into a tissue processor for processing through to wax.

  • Gross section should not be more than 4 mm thick/wide. When processing on a short protocol, the gross section must be thinner or the reagent will not completely penetrate the section.

  • Gross section should not be too thick that it touches both the top and bottom of the tissue processing cassette.

  • This surgical specimen of stomach has been fixed in formalin. Slices about 4mm thick will now be taken from appropriate areas and placed in the labelled cassettes for processing.

Tissue Processing

  • Tissue processing can be performed manually (hand processing) or via an automated tissue processing machine (multiple specimens at a time, more convenient and efficient).

  • Two main types of tissue processors:

    • Modern enclosed tissue processor (automated):

      • Fluid-transfer types where specimens are held in a single process chamber or retort and fluids are pumped in and out as required.

    • "Dip and dunk" processor (manual handling)

      • tissue-transfer machines where specimens are transferred from container to container to be processed

Steps in Sample Preparation for Histology

  • Take tissues from body -> Process tissue in the lab (Fixation -> Dehydration, Clearing, Infiltration & Embedding -> Sectioning & Staining) -> Microscopic examination of section/specimen -> Diagnosis of disease.

  • After sectioning, tissue sections in wax are mounted to microscope slides before staining

Analogy to Fixation

  • Slicing Chicken (raw versus cooked) or laminating a document…

Why Fixation?

  • Fresh tissue (soft, delicate, easily distorted & damaged) => Requires support for it to be cut -> Very thin high quality sections mounted on glass slides -> Stained to demonstrate normal & abnormal structures -> Microscopic analysis of cells & tissues

  • A fresh, unfixed specimen after surgical removal. To prevent degeneration or drying-out the specimen should be fixed as soon as possible.

Tissue Fixation

  • Fixation preserves tissue to keep the cellular morphology intact to ensure that the specimen is diagnostic

  • Stabilizes tissue proteins to prevent further changes, such as decay or putrefaction and autolysis (enzyme attack)

  • Fixation should be carried out as soon as possible after removal of the tissues (in the case of surgical pathology) or soon after death (with autopsy) to prevent autolysis

  • The fixative volume should be 15 to 20 times greater than tissue volume

Fixation Methods

  • Physical methods

    • Heating

      • Applications:

        • Cell smears

        • Accelerate chemical fixation

    • Freezing

      • Applications:

        • Delicate antigens, non suitable for harsh protocols

    • Microwave

      • Applications:

        • Primary microwave stabilization

        • Microwave-assisted fixation

  • Chemical methods

    • Coagulant fixatives

      • Alcohols

      • Acetone

      • Acetic acid

      • Picrates

      • Mercurials (e.g. mercuric chloride)

    • Cross-linking fixatives

      • Aldehydes

    • Oxidizing agents (osmium tetroxide, potassium dichromate, potassium permanganate, chromic acid,…)

Common Fixatives

  • The most popular fixing agent is 4% paraformaldehyde or 10% formalin, usually in the form of a phosphate-buffered solution (often referred to as “formalin”)

Terminology

  • FFPE tissue = Formalin-fixed paraffin embedded

Terminology

  • Denature: Destroy the characteristic properties of (a protein or other biological macromolecule) by heat, acidity, or other effect which disrupts its molecular conformation

  • Coagulant: Coagulants are substances which cause particles in a liquid to curdle and clot together. Coagulants carry the opposite charge to the particles and therefore cause the charge to 'destabilise' when added to the water; resulting in the particles clinging together.

Mechanism of Chemical Fixation

  • Dehydration/Coagulation: Destroys protein tertiary structure by displacing water, causing protein precipitation (ethanol, methanol, and acetone)

  • Denaturation: Denature proteins and nucleic acids through pH changes/salt formation (acetic acid, trichloroacetic acid, mercuric chloride, and zinc acetate)

  • Cross-linking/Additive: Add covalent reactive groups to induce cross-links between proteins, individual protein moieties, within nucleic acids, and between nucleic acids (e.g. formaldehyde and glutaraldehyde)

  • Compound fixatives: Mixtures of reagents e.g. alcoholic formalin fix tissues by adding covalent hydroxymethyl groups and cross-links as well as by coagulation and dehydration.

Terminology (Formaldehyde, Formalin, Paraformaldehyde)

  • Formaldehyde = water soluble gas

  • Formalin = Formaldehyde in water

  • 10% Formalin or 4% formaldehyde = 1:10 dilution of formaldehyde in water [100% formalin contains 40% formaldehyde]

  • Paraformaldehyde = polymerized formaldehyde (powder form)

Chemical Fixative: Aldehydes

  • Formaldehyde – most widely fixative used in histopathology preserves the peptides of cellular proteins→ useful as a general-purpose fixative

  • Cross-links are formed between protein molecules and aldehydes

  • Glutaraldehyde is more effective at forming cross-links than formaldehyde → effectively preserves the ultrastructure* of cells and is the fixative of choice for electron microscopy

  • Glutaraldehyde-fixed tissues stain poorly with conventional dye-staining methods

    • Ultrastructure*: Fine structure, especially within a cell, that can be seen only with the high magnification obtainable with an electron microscope

  • The reaction between aldehydes and proteins is pH-dependent (faster at high pH)

  • The reaction with formaldehyde is reversible with an excess of water within 24 hours

  • Formaldehyde (CH2OCH_2O) is the only gaseous aldehyde and is dissolved in water to saturation at 37% – 40% w/v. This solution is generally referred to as “formalin” or “concentrated formaldehyde solution”

  • The reaction with glutaraldehyde is rapid and irreversible and is characterised by the formation of many cross-links

Formaldehyde

  • Formaldehyde is the most commonly used fixative in histopathology, despite concerns about its toxicity

  • It is irritant, corrosive and may cause allergic sensitization

  • Known human carcinogen

  • Be aware of the hazards involved!

  • 4% Formaldehyde solution (or 10% Formalin) at pH 6.8 - 7.2 = most commonly-used fixative

Chemical Fixative: Oxidising Agents

  • Osmium tetroxide, potassium dichromate and potassium permanganate

  • Mechanism of cross-link with proteins is less known

  • Osmium tetroxide is commonly used in processing samples for transmission electron microscopy

  • Osmium tetroxide is a good fixative and excellent stain for lipids in membranous structures and vesicles

Tissue Damage During Fixation

  • Fixation damages proteins

  • Optimise fixation for enzyme histochemistry, immunocytochemistry and electron microscopy in order to preserve antigenic sites

  • Balance: retain biological activity vs good morphological preservation

Different Fixative Effects on Cellular Morphology

  • A paraffin section from the mucosa of small intestine that has been fixed in 95% ethanol, a denaturing fixative. While nuclear preservation is fair there is substantial shrinkage of cytoplasmic and extracellular elements.

  • A paraffin section of the mucosa of small intestine that has been fixed in neutral buffered formalin, a cross- linking fixative. Nuclear and cytoplasmic preservation is satisfactory but some cellular shrinkage is present

  • Importance of comparing images at the same magnification to ensure valid and fair comparison

Physical Fixative: Microwave Fixation/ Stabilisation

  • Rapid fixation

  • Valuable for processing urgent samples (eg. cardiac biopsies following heart transplantation) as patient therapy depends on rapid interpretation

Other Types of Physical Fixation

  • Freeze-drying allows rapid study of specimen (e.g. during a surgical procedure)

  • Steps in specimen preparation:

    • Freeze the tissue

    • Dehydrate the sample under vacuum

    • Embed the dehydrated sample

Importance of Proper Fixation

  • Histological Quality: Proper fixation is essential for high-quality tissue sections.

  • Immunohistochemistry: Antigen retrieval* may be needed to unmask epitopes.

  • Research and Diagnosis: Accurate results in research and clinical pathology depend on effective fixation.
    *Fixatives like formaldehyde create protein cross-links, masking antigenic sites (epitopes). Antigen retrieval breaks these cross-links to unmask the sites, improves antibody binding to specific targets, leading to more accurate staining and better diagnostic results. Two types of antigen retrieval: (1) Heat-induced epitope retrieval (2) Enzyme-induced epitope retrieval.

Factors Affecting Fixation Quality

  • Buffers and pH (hydrogen ion concentration)

    • As near the biochemical optimum as possible (adjust pH to 6-8 with suitable buffer)

  • Duration of fixation

    • Usually 2-6 hours but can be longer (e.g. 24 hours); prolonged fixation may cause tissue shrinkage/degradation, inhibit enzyme activity, destroy antigen site for IHC

  • Specimen dimension

    • Gross tissue should be of even thickness (no more than 3 to 4 mm)

  • Temperature of fixation

    • Room temperature or heated fixation (for light microscopy) allows quicker fixation/penetration of fixative vs cold temperature (usually for electron microscopy)

  • Concentration of fixative

    • Optimise fixation concentration (influenced by cost, effectiveness and solubility) and be mindful of masking of antigenic sites

  • Penetration rate

    • Varies among fixatives; generally slow so tissue blocks should be small or thin

  • Osmolality of fixatives and ionic composition

    • Ideally isotonic with tissues in their living state (slightly hypertonic solutions work best for electron microscopy)

  • Volume ratio

    • Adequate fixing agent at minimum 15-20:1 fixative to tissue ratio

Specimen Dimension

  • A specimen (grossed) should not be more than 4 mm thick (3 mm- thick slice should provide excellent fixation and processing)

  • Note that the specimen cavity (depth) in a standard processing cassette is 5 mm deep

  • Note the difference between ‘Grossed tissue’ specimen 3-4 mm thick (Pre-fixation and pre-embedding) vs section embedded in wax (Post-fixation and post-embedding) “Thinly-sliced tissues”-> thin sections 5-10 um thick for staining/microscope visualization)

Fixation Artifacts

  • Fixation aims to prevent artefacts and maintain good tissue architecture.

  • However, it should be understood fixation methods may cause:

    • Volume changes

    • Diffusion of unfixed material to give a false localisation

    • Formalin pigment formation under acid conditions

    • Autofluorescence
      Artefact = something observed in a scientific investigation or experiment that is not naturally present but occurs as a result of the preparative or investigative procedure.

Inadequate Fixation Time (Under-Fixed)

  • Limit cold ischemia* time to <1hr. The more time that elapses between interruption of the blood supply and fixation (cold-ischemic time), the more postmortem changes can be demonstrated microscopically.

  • Tissue should be placed in fixative upon collection.

  • Fixation time 6-72 hours; 24 hrs in fixative is optimal.

  • Ratio of formalin to tissue for optimal penetration is 10-20 to 1.
    *Cold ischemia time refers to the duration between the interruption of blood supply to a tissue or organ and its preservation by fixation or freezing. During this period, the tissue is typically kept at a low temperature (on ice or refrigerated) to slow down enzymatic activity and minimize degradation.

  • Under-fixation causes significantly more staining problems than over-fixation. Incomplete formalin fixation can result in alcohol fixation of the remaining tissue, producing inconsistent staining across a stained tissue section.

Implications of Over-Fixation

  1. Antigen Masking: Excessive cross-linking of proteins masks antigenic sites, reducing antibody binding efficiency in immunohistochemistry (IHC).

  2. Poor Staining Quality: Over-fixation can lead to diminished staining intensity and inconsistent results.

  3. Tissue Hardening: Tissues become overly rigid, making microtomy (sectioning) challenging.

  4. Loss of Enzyme Activity: Enzymatic sites may be permanently damaged, hindering enzyme histochemistry studies.

  5. Prolonged Processing Times: Over-fixed tissues take longer to process and require additional steps, such as antigen retrieval, to restore staining compatibility.

Implications of Under-Fixation

  1. Inadequate Preservation: Cellular and tissue structures degrade due to incomplete stabilization of proteins.

  2. Autolysis and Decay: Insufficient fixation allows enzymes within the tissue to break down cells, leading to autolysis and compromised morphology.

  3. Poor Staining and Diagnostic Value: Uneven or incomplete fixation results in inconsistent staining and unreliable diagnostic interpretation.

  4. Increased Staining Artifacts: Unfixed regions may show artifacts, such as non-specific binding of stains or uneven dye penetration.

  5. Soft Tissue Consistency: Tissue remains too soft, making sectioning difficult and leading to tears or distortions during microtomy.

Choice of Fixatives

  • Nature of the Specimen

    • Tissue Type: Different tissues may require specific fixatives.

    • Size and Thickness: Larger specimens may need special considerations.

  • Purpose of Fixation

    • Routine Histology: Formalin for general tissue preservation.

    • Electron Microscopy: Osmium tetroxide for ultrastructural details.

    • Molecular Studies: Alcohol-based fixatives or snap-freezing to preserve DNA/RNA

    • Immunohistochemistry (IHC): Consider preservation of protein epitopes. Formalin-fixed tissues are common for IHC.

  • Downstream Applications

    • Consider the techniques or analyses that follow fixation.

    • Some fixatives may interfere with specific assays.

  • Time Constraints

    • Fixatives vary in speed; choose based on available time.

  • Toxicity and Handling

    • Consider toxicity, especially with highly toxic fixatives. Safety precautions need to be in place.

  • Expertise and Laboratory Protocols

    • Experience and established protocols influence fixative selection.

  • Cost Considerations

    • Cost of fixatives and reagents may impact choices.

Other Factors Affecting Fixation

  • The period of ischaemia* experienced by the tissue during the collection process will adversely effect tissue integrity (e.g.):

    • Surgical clamping

    • Post-mortem tissue
      *Ischaemia: an inadequate supply of blood to an organ or part, as from an obstructed blood flow

Penetration Rate of Fixatives

  • The effectiveness of a fixative will depend on how well it penetrates into tissue

  • The process is slow so tissue blocks should be small or thin

  • Medawar found that the depth penetrated (d) was proportional to the square root of the time (t):

    • d=Ktd = K\sqrt{t}

      • Where K is the coefficient of diffusibility at 1 hour in mm that the fixative has diffused into tissue

  • Generally measured on liver or gelatin pessaries (gels)

  • Slow rates of diffusion and reaction give rise to various zones in tissues which have been fixed to different degrees

Maths

  • Medawar found that the depth penetrated (d) was proportional to the square root of the time (t):

    • d=Ktd = K\sqrt{t}

  • Let’s assume that for 10% formalin K = 1 where K is the coefficient of diffusibility at 1 hour in mm that the fixative has diffused into tissue.

  • How long will it take 10% formalin to penetrate the centre of a 10 mm thick specimen? (Calculate t)

    • t = 25 hours (5 = 1√t√t; therefore √t√t = 5; t = 25)

Factors Affecting Tissue Processing

  • Size/Thickness

  • Lipid content

  • Density

Tissue Dehydration

  • Removes water from tissue with alcohol

  • Why? Wet fixed tissues (in aqueous solutions) cannot be directly infiltrated with paraffin wax. Through the dehydration process, all the fixating solution and tissue fluid is removed.

  • Alcohol is used to dehydrate the tissue to make the tissue miscible (capable of being mixed specifically) with wax.

  • Gradual removal of water from tissue is preferred, so the alcohols typically begin with 70% and finish with several changes of 100% alcohol.

  • Other dehydrants can be used, but major disadvantages (e.g. Acetone acts rapidly, but a fire hazard; Dioxane can be used without clearing, but has toxic fumes).

Tissue Clearing

  • Remove dehydrants (alcohols) and permit tissue infiltration with paraffin wax.

  • Replace the dehydrant with a substance (can be xylene, xylene substitutes (Citroclear) or isopropyl alcohol) that will be miscible with the embedding medium (paraffin)

  • Xylene is commonly used to clear tissue so it can be impregnated with paraffin wax

  • A couple changes in the clearing agent are sufficient

Other Clearing Agents

  • Toluene works well, but is 3 times more expensive than xylene

  • Chloroform used to be used, but is a health hazard, and is slow

  • Methyl salicylate smells nice but is rarely used because it is expensive

Paraffin Wax Infiltration

  • Displaces clearing agent in tissue with paraffin wax, in preparation for paraffin wax embedding

  • Tissue is infiltrated with liquid wax (60°C) and then cool to 20°C where it solidifies to a consistency that allows sections to be consistently cut with a microtome

Tissue Embedding

  • Process by which tissues are surrounded by a medium such as wax, agar, or gelatin which when solidified will provide sufficient external support during sectioning

  • Embedding is important in preserving tissue morphology and giving the tissue support during sectioning/microtomy

  • This "embedding" process is very important and tissues must be aligned, or oriented, properly in the block of paraffin to facilitate sectioning

Sectioning & Mounting on Slides

  • Once the tissues have been embedded, they must be cut into sections (4-10 μm) that can be placed on a slide

  • This is done with a microtome - a knife/blade with a mechanism for advancing a paraffin block standard distances across it

Staining

  • The staining process makes use of a variety of dyes that have been chosen for their ability to stain various cellular components of tissue

  • The routine stain is that of Haematoxylin & Eosin (H & E)

  • Before any staining can be done, the slides are "deparaffinized” (“dewax” in practical) by running them through xylenes (or substitutes), then to alcohols, then to water

Microscopic Examination of Histological Specimen

  • Histology: Prepare thin slices (to allow light to penetrate) of tissues (5-10 μm thick)

  • Stain sections with dye

  • Higher magnification power/objectives ≠ more information about a specimen!

  • Understand the big-picture first..

  • Always start with the lowest magnification objective (large field vision)

  • Beware of sectioning and staining artefact (something you see on the specimen which is not a normal component of that tissue/organ)

  • Sectioning plane is also important (transverse/longitudinal)..

Histochemical Staining

  • Histochemistry is used to localize and identify substances in a tissue by means of chemical reactions

  • Substances can be ions (calcium, iron, copper, zinc, etc.), proteins (primarily enzymes), carbohydrates and lipids

  • Staining is used to highlight important features of the tissue as well as to enhance the tissue contrast

Histochemical Staining - Basic and Acid Dyes

  • Basic dyes are cationic (+ve charged). They form salts with tissue anions (components that carry a net negative (-ve) charge, especially the phosphate groups of nucleic acids)

  • Acid dyes are anionic (-ve charged). They form salts with cationic (+ve charged) groups in cells and tissues, particularly the ionized amino groups of proteins

  • Nuclei (-ve) are basophilic (loves basic (+ve) stains)

  • The cytoplasm (+ve) is usually acidophilic (loves acidic (-ve) stains)

Reminder

  • Acidic dyes (-) stain basic (+) components of the cell (i.e. cytoplasmic (basic) components are acidophilic)

  • Basic dyes (+) stain acidic (-) components of the cell (i.e. nucleic acid (acidic) components of the cell are basophilic).

  • Examples:

    • Most proteins in the cytoplasm are basic. This includes cytoplasmic filaments in muscle cells, intracellular membranes, and extracellular fibres.

    • DNA in nucleus

    • RNA in ribosomes and in the rough endoplasmic reticulum.

    • Carbohydrates in cartilage

Haematoxylin and Eosin (H & E) Staining

  • The most widely used histological stain (quick, relatively inexpensive)

  • Simple method to clearly demonstrate a large number of different tissue structures.

  • Haematoxylin:

    • Cell nuclei stains purplish-blue/black with good intra-nuclear detail

  • Eosin:

    • Stains cytoplasm (amino groups of proteins) and connective tissue with varying intensities of pink, orange and red

Cationic Dyes

  • Cationic dyes bind to negatively charged cellular structures

  • Negatively charged groups of molecules within cells:

    • half-sulfate ester groups of glycoconjugates (SO4SO_4 -)

    • phosphates of nucleic acids (PO4PO_4 -)

    • carboxylate groups of glycoconjugates and proteins (COO-)

  • Ionization ability of molecules is pH-dependent (from strongest to lowest ionization ability):

    • Sulphuric acid groups (SO<em>4SO<em>4 -) > Phosphoric acid (PO</em>4PO</em>4 -) groups > Carboxyl groups (COO-)

  • Therefore pH influences staining properties of cationic dyes (i.e. Highly ionized sulfate or phosphate groups will have stronger binding to cationic dyes compared to carboxyl groups).
    *Ionization, is the process where electrically neutral atoms or molecules are converted to electrically charged atoms or molecules (ions).

Haematoxylin

  • Natural dye extracted from logwood of Haematoxylon Campechianum tree

  • One of the best nuclear stain

  • Haematoxylin on itself is not an active dye

  • Its major oxidative product (haematein) is responsible for the colour.

Haematein

  • Haematein (oxidized product of haematoxylin) is responsible for dye colour

  • Haematein can be produced by:

    • Natural oxidation (‘ripening’) by exposure to air/sunlight (slow process; 3-4 months, longer shelf life)

    • Chemical oxidation using sodium iodate or mercuric oxide (have a