BASIC STAINING TECHNIQUES LECTURE 1

OBJECTIVES: At the completion of this section the student will be able to: Know the steps involved in tissue processing and discuss the different types of microscopy. lnterprete the cytological significance of differential staining produced by hematoxylin and eosin (H&E). Recognize and identify cells in microscopic tissue preparations. Understand that morphology reflects the function of cells.

Methods Histochemistry Cytochemistry Immunocytochemistry Organ & tissue culture Differential centrifugation Specialized microscopic techniques

Histology is the study of tissue sectioned as a thin slice, using a microtome. It can be described as microscopic anatomy. Histology is an essential tool of biology.

Histopathology, the microscopic study of diseased tissue, is an important tool of anatomical pathology since accurate diagnosis of cancer and other diseases usually requires histopathological examination of samples.

Histochemistry refers to the science of using chemical reactions between laboratory chemicals and components within tissue. antibodies are used to specifically visualise proteins, carbohydrates and lipids: this is called immunohistochemistry.

Histochemistry and Cytochemistry Tissue Preparation Methods for the observation of - living cells - dead cells Example- micro-organism like bacteria, virus, fungi etc

Living Cells Cells from tissue cultures and unicellular organisms observed with a phase-contrast microscope

Dead cells Sample sources Autopsy Biopsy Blood samples

Stages of the Histology Technique Light microscopy Sampling Fixation Dehydration Clearing Infiltration (impregnation) Sectioning Staining Mounting Embedding

Aim – is to provide a representative specimen Sampling Aim – is to provide a representative specimen

Fixation Aim – to preserve the cells with the “least alteration” possible from the living state Use of substances that coagulate the protoplasm Simple – formaldehyde, alcohol, acetic acid, osmic acid, picric acid Complex – Bouin’s fluid, Zenker’s fluid, special mixtures Some organs needs fixation even before the macroscopic preparation takes place 10 % formaldehyde is the cheapest and easiest to keep fixative solution

Dehydration Aim – to remove water from the sample to allow paraffin impregnation Done by passing tissue sample through gradually increasing concentrations of alcohol

Clearing Aim – to take the alcohol out of the sample to allow paraffin to impregnate the tissue The tissue is immersed in xylene (xylol) that is miscible both in the dehydrating and the embedding agent The name of the process come from the clear appearance the sample gets Benzene, cedarwood oil, chloroform and others can also be used

Infiltration Aim – to incrust the sample inside a material that allow 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 The embedded specimen in the plastic frame can be (and should be) safely conserved for many years It is very important to properly label each one of the plastic frames

During this 12 to 16 hour process, paraffin wax will replace the water: soft, moist tissues are turned into a hard paraffin block, which is then placed in a mould containing more molten wax (embedded) and allowed to cool and harden. Embedding can also be accomplished using frozen, non-fixed tissue in a freezing medium. This freezing medium is liquid at room temperature but when cooled will solidify.

Microtome A microtome is a mechanical instrument used to cut biological specimens into very thin segments for microscopic examination.

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 the stain that is going to be used The section is transferred to a glass slide

Sectioning Tissue block is cut into 1~20μm thick sections by microtome, and laid out on the surface of warm water and are then adhered to gel-coated slides.

Staining Aim – to add color to the structures of the sample in order to differentiate them There are several phases of staining Every stain has a different procedure (phases, time, extra procedures, etc.) Special stains color different structures Most common, easiest to use & cheapest stain is hematoxylin and eosin (H and E)

Dehydration and clearing Before After

Mounting Aim – to put the final sample in a medium where it is protected and can also be observed under the microscope The sample is dehydrated again A drop of mounting agent with a similar refractive index to that of glass is placed on the glass slide, and covered with a glass coverslip is added Mounting medium - DPX - Canada Balsam diluted in xylene (xylol)

(Important during surgical biopsy diagnosis) Freeze Drying Method (Important during surgical biopsy diagnosis) Aim – to quickly prepare a glass slide for rapid study of specimens during a surgical procedure Steps involved in specimen preparation: Freeze the tissue Dehydrate the sample under a vacuum Embed the dehydrated sample The quality of the section by this method is poor, and a regular histological technique should followed at the end of surgery.

***immersion of tissues in xylene dissolves the tissue lipids, which is undesirable effect when these compounds are to be studied. To avoid loss of lipids, a freezing microtome has been devised in which the tissues are hardened at low temperatures to provide rigidity necessary to permit sectioning. The freezing microtome is a more elaborate and efficient successor of the cryostat.***

Cell Smears Cell smears are a form of histological preparation that does not require sectioning. Smears can be made for example of the blood or bone marrow. Smears are also common for swabs or scrapings of epithelial cells (e.g. from the oral cavity, cervix uteri).

MATERIALS - sterilized lancet or needle - 20 clean microscope slides and coverslips - Canada balsam or other medium for permanent preparations - 95% ethyl or methyl alcohol - distilled water - Giemsa stain - low containers (you can make them with aluminum sheet also) - microscope which magnifies 200 times at least

TAKING THE BLOOD MAKING THE SMEAR FIXING STAINING CHECKING COVER-SLIPPING OBSERVATION

MAKING THE SMEAR

FIXING fixing technique consists of dipping the smear in a vessel containing 95% ethyl or methyl alcohol for 3-5 minutes.

STAINING To be able to observe and recognize the different kinds of leukocyte, you must stain them. For this purpose, normally Giemsa stain is used. It is a mixture of stains, based on methylene blue and eosin

Electron Microscopy

Stages of the Histology Technique Electron microscopy Small samples (1 mm3 or less) Tissue must be fresh Double fixation (glutaraldehyde for proteins plus osmium tetroxide for lipids [osmium also stains]) Quick dehydration and clearing Embedding in plastic resins (Epon or Araldite) Cut with ultramicrotome (glass or diamond knifes) produces 30 – 50 nm thick specimens Mounting in copper grids Stain with heavy metals

Tissue Fixation in Electron Microscopy Same principles as in Light Microscopy with the following differences: Double fixation in glutaraldehyde and osmium tetroxide Ultra-thin sections (< 90nm thick) from plastic embedded blocks. Thus, all the original EM pictures are black-white images.

TEST YOURSELF The agents in which procedure are intended to stabilize tissue structure by coagulating proteins and promoting cross-linking in? Clearing Dehydration Embedding Fixation

Stains Acid dyes stain basic structures: (proteins, membranes, cytoplasm). The commonest acid dye is eosin; it is pink Basic dyes stain acid structures: Nucleic acids (RNA, DNA, rER, nuclei). The commonest basic dye is hematoxylin; it is blue NOTE: tissues that stain with basic dyes are termed basophilic; tissues that stain with acid dyes are termed acidophilic Nuclei are basophilic, since they are mainly made of DNA Cytoplasm is acidophil, since most proteins are basic in nature

Useful stains in histology Regular Stains: Hematoxylin & Eosin (H&E) Trichromes (differentiate intercellular structures) Iron hematoxylin (for iron-containing cells, like muscle & red blood cells) Specific stains: Proteoglycans (PAS, cresyl violet, toluidine blue, methylene blue) Neurons (Nissl, Ag, Osmium, cresyl violet) Elastic fibers (orcein or resorcein) Reticular fibers (silver stain) Blood cells (Romanovsky stains) Polysaccharides (PAS)

Haematoxylin-eosin staining H&E is the most common dye used in the study of histology Hematoxylin stains acidic structures (nucleic acids, nuclei, rER) blue Eosin stains basic structures (proteins, membranes) pink

Structures that are not readily stained by hematoxylin or eosin, are called neutrophilic structures.

Which of the following statements is false about the rough endoplasmic reticulum(RER)? (A) Prominent in cells specialized for protein secretion (B) Presence of polyribosomes confers basophilia (C) Presence of polyribosomes confers acidophilia in the cell (D) This organelle can be viewed with a light microscope

Trichrome stain Uses three dyes to differentiate intercellular structures Particularly helpful in highlighting red blood cells within blood vessels

Trichrome stain Gomori trichrome Mallory’s trichrome Masson’s trichrome Red keratin and muscle fibers, Blue or green collagen and bone, Light red or pink cytoplasm, and Dark brown to black cell nuclei

Trichrome stain Note the blue nuclei, the pink/purple cytoplasm, the red blood cells and the tuquoise material that is non-cellular.

Trichrome stain Note: Blue nuclei Pink color in cytoplasm Deep red of blood cells

Diff btw H&E and Trichrome stain

Iron-haematoxylin Highlights tissue and cells that contain iron, such as muscle & red blood cells

Specific stains for proteoglycans Periodic acid-Schiff (PAS) reaction ALSO USEFUL FOR THE IDENTIFICATION OF POLYSACCHARIDES (Glycogen) Cresyl violet Toluidine blue Methylene blue

Periodic Acid Schiff (PAS) staining PAS reaction is a method to demonstrate glycogen-like substances. Periodic acid oxidizes the glycol groups in the glucose residues into aldehyde groups, which then react with Schiff’s reagent, producing an insoluble compound with a reddish purple color. PAS reaction in a hepatocyte PAS reaction in mucosa of intestine

Specific stains used for NEURONS Nissl (a specialized stain for rER of neurons) Silver & Gold (for fibers & cytoskeletal elements) Osmic acid (for myelin, a lipid) Cresyl violet (proteoglycans)

A specialized stain for rough endoplasmic reticulum in neurons Nissl staining A specialized stain for rough endoplasmic reticulum in neurons

Neurofilaments stained Silver Staining Reticular fibres Neurofilaments stained with silver salts Silver ions are deposited on the structures and reduced to silver particles, showing brown to black color, (argyrophilic). Reticular fibers cannot be well seen with H&E staining

Reticular stain

Cresyl violet Highlights proteoglycans Most commonly used for staining nervous tissue This is a violet/purple stain

Elastin stains elastic fibers black

Verhoff stain for elastin Elastic stains such as Verhoff's stain is more specialized, but can be useful to determine changes in vascular or respiratory function due to compromised elastin content. In these images, the elastic fiber content of small arteries is demonstrated

Romanovsky Stain for Blood Cells Note the following: Reddish-brown cytoplasm of red blood cells Large (white) cell with deep blue granules White blood cells are most readily identified with the use of Romanovsky – type stains E g GIEMSA Romanovsky stains (Giemsa, Wrights, etc.) are useful for highlighting various types of granules present in developing and mature white blood cells.

Metachromasia Phenomenon where a certain dye shows structures in a different color from that of the dye. The granules in cytoplasm of mast cells are shown in purple color when stained by toluidine blue, a blue dye.

Toludine blue

Osmic acid staining

****The effect of osmium tetraoxide is to preserve and stain lipids and proteins.*****

Oil Red O Oil Red O is used to stain lipids a red-orange color in unfixed frozen sections.

Methylene blue staining Periodic acid-schiff(PAS) reaction . Q1. which technique is most commonly used to locate glycogen in cells? Methylene blue staining Periodic acid-schiff(PAS) reaction Enzyme histochemistry Q 2. frozen sectioning may be required to avoid the removal of which target substance when preparing tissues for paraffin sectioning? Basic proteins Lipids Enzymes carbohydrates Q 1

ARTIFACTS

Pre-histology These are features and structures that have being introduced prior to the collection of the tissues. A common example of these include: ink from tattoos and freckles (melanin) in skin samples. Post-histology Artifacts can result from tissue processing. Processing commonly lead to changes like shrinkage, color changes in different tissues types and alterations of the structures in the tissue. Because these are caused in a laboratory the majority of post histology artifacts can be avoided or removed after being discovered.

Artifacts (not natural occurences) Caused by a bad histological technique Autolysis Poor sampling Shrinkage Folds Stain precipitation and dust Defects in the knife The main cause of artifacts is poor fixation Some tissues usually present artifacts, & wrong knowledge has been derived from them

Suprarenal gland showing with autolysis

Shrinkage and rupture

Shrinkage

Folds

Breaking and notches

Knife marks

Edge damage

Poor fixation

Dust