Methods to study HistologyKrishna T

Cell Tissue Organ Organ system Homeostasis

Histology

How to get tissues for study Steps in tissue preparation Fresh tissues from the body 1. fixation

◦ Formalin ( 10% formaldehyde)◦ Osmium tetroxide for EM◦ Mechanism - Forms cross links with proteins (Lysine)

2. Embedding – gives support for tissue slicing◦ Paraffin or plastic resin

3. Washing & dehydration (dehydration by graded alcohols in ascending order)

4. clearing – to remove paraffin & alcohol◦ By xylol or tulol

5. block making

How to get the histology slides?

6. section cutting – 5-10μ thick sections with microtome 7. mounting – on glass slide ( adhesive – albumin) 8. clearing – xylol / tulol 9. rehydrate – alcohols in descending order Staining

◦ nuclear stain – Hematoxylin ( basic stain & water soluble)◦ counter stain – Eosin ( less water soluble but soluble in

alcohol) – dehydrate in ascending order 10. Clearing – xylol / tulol 11.Mounting medium – cover glass

How to get the histology slides?

Staining – routine stain – H&E◦ Some structures are seen/ preserved (large molecules like

nucleoproteins, cytoskeleton proteins, ECM proteins- collagen, membrane proteins)

◦ some are not seen/lost (small molecules -t-RNA, large molecules like glycogen & Proteioglycans are dissolved, )during the fixation/staining process

Special fixatives to retain membrane ( phospholipids)◦ Permanganate & osmium – for EM

For Elastic fibers – Orcein/ Resorcin – Fuscin For reticular fibers – Silver impregnation Histochemistry & Cytochemistry

◦ Specific binding of dye with particular molecule◦ Fluorescent dye labeled antibody to cell component

Enzyme activity Autoradiography – radio isotopes tagged with precursors of a

molecule molecule incorporated into cell/ tissue before fixation

Special situations

Basis of staining

ACIDIC DYES BASIC DYES

Eosin Hematoxylin /Methylene blue

Carry net negative charge

Carry net positive charge

React/bind with cationic components of the cell/tissue

With anionic components of cell/tissue

Less specific (as compared with basic dyes)

Highly pH specific

Acidophilic / Eosinophilic (cytoplasmic filaments, intracellular membranous components, extracellular fibers)

Basophilic substances ( Po4 of Nucleic acids, So4 of MPS, CO proteins)

Mostly resembles basic dye but it is a mordant (helps to form links between tissue fragment & the dye)

It will not dissociate in sequential staining process unlike other basic dyes

What is special about Hematoxylin?

What is it ? Absorb certain wavelength of light and emit different wavelength

Why Metachomasia ? Polyanions of tissues bind with dye molecules result in polymer or dimers of dye molecules appear as different color rather than expected ( methylene blue gives red or purple color)

What are metachromatic substances? Ionized So4, Po4 of cartilage

Where you find it? Mast cell granules (heparin) & rER of Plasma cells

Metachomasia

Special stain PAS positive substances

Carbohydrate (glycogen) or carbohydrate rich molecules, Basement membrane, reticular fibers

Periodic acid cleaves bond between carbon atoms form aldehyde group

Aldehyde binds with Schiff to produce magenta or pink color

PAS =Periodic Acid Schiff

Acid hydrolyses or cleaves proteins from deoxyribose of DNA leads to opening of sugar group & formation of aldehyde

Schiff binds and gives magenta color to aldehyde

Can be useful to quantify amount of DNA ( by using spectrophotmetry of Feulgen stained tissue)

Feulgen stain for Nuclear Proteins

Why RNA cannot be stained by Feulgen?

For the confirmation of specific substances Pretreatment of sections with specific

enzymes Diastase/amylase for glycogen DNA ase for DNA

Enzymatic digestion

Localization of enzymatic activity in tissues Best fixation – mild aldehyde ( formalin) Basis – localized reaction production of

enzyme activity Used for acid & alkaline phosphatase, ATP

ases AB (substrate) + T (trap) AT

( reaction product) + B (Hydrolyzed component of substrate)

Enzyme Histochemistry

enzyme

Antibody ( Immunoglobulin) conjugated with fluorescent dye( most common is Fluorescein) + Antigen ( foreign protein)

Fluorescein absorbs UV light and emits green fluorescence can be seen under Fluorescent microscope (IF- Immuno Fluorescence)

Example :- actin (Antigen) of Rat infected to Rabbit blood of Rabbit ( have poly - clonal antibodies for Rat’s actin/ anti rat actin antibodies) bind with Fluorescent dye

Immuno Histo Chemistry (IHC)

Specific antigen (actin of rat)

Monoclonal Antibodies

Multiple Myeloma pts.

Monoclonal B ells

Hybridoma cells ↓

Single specific type of antibodies (Monoclonal) ( against Actin)

B lymphocytes ofImmunized rabbit

↓

Diagnosis of tumors(tumor markers) & Infections( HIV, Infectious Mononucleosis)

Classify sub – types (B -cell and T- cell lymphomas)

Treatment – Anti-TNF-α antibodies in inflammatory disorders

Clinical Significance of Monoclonal Antibodies

Immuno -fluorescence ◦ Direct (one step, less sensitive)

& ◦ Indirect ( more sensitive, Expensive, labor

intensive, can’t easily run in automated) methods Immunoperoxidase method

◦ Enzyme is used ( horse raddish peroxidase) to color colorless substrate into colored insoluble product

Immunological Methods

Other Methods

Hybridization: for localizing mRNA/DNA (NA)

In Situ Hybridization: Binding ( Probe + NA) in cell/tissue

FISH: If Fluorochrome is used in Hybridization technique

Autoradiography: by tagging the precursor molecules (Amino acids) followed by synthesis of large molecules (NA) localize the particular tagged molecule

Microscopy

Resolution/ Resolving power (RP): the distance by which two objects must be separated to be seen as two objects◦ RP of

Unaided Human retina : 0.2 mm Light Microscope (LM) : 0.2 μ Electro Microscope (EM) : 1.0 nm

LM: we see only two dimensional pictures, orientation of cut gives different patterns

Artifacts: error in preparation process

orientation of cut

Three dimensional picture

How you get it?

Types & Advantages of Microscopes

1. Phase contrast M: ◦ can see live (unstained) tissue◦ Light passing thru denser tissue of higher

refractory index out of phase from the rest look darker

◦ Uses : identify cells in tissue cultures◦ Modification: Interference M: quantification of

tissue masses helps in study of surface properties of cells

What happens to the tissues during routine staining process?

Types & Advantages of Microscopes 2. dark Field M: special condenser illuminates

specimen with strong oblique light◦ Uses:

In auto radiography Study crystals in urine Study microbes- slender spirochetes ( *Treponema pallidum)

3. Fluorescent M: emits light in visible range when exposed to UV light◦ Technique: filters are used between light source &

specimen◦ Naturally fluorescent substances: Vitamin “A”, Neuro-

transmitters◦ Uses

Tracing pathways of nerve fibers, To detect growth markers of mineralized tissues

*What is the disease caused by this bug?

Types & Advantages of Microscopes

4. Confocal scanning M: ◦ Conjugate with focal point of lens◦ Computer software reconstitutes the image from the data◦ Major difference from LM: addition of detector

aperture (pin hole)◦ Uses: can see 3D pictures

5. ultra violet M: ◦ Depends on absorption of UVL by specimen◦ Results are recorded photographically (can’t be seen

directly – why?)◦ Uses

Study of nitrogen bases ( in NA) Study amount of DNA/RNA in cells *Clinically helps in study

of ploidy in tumors

Highly aneuploid tumor What is its Significance ?

Types & Advantages of Microscopes

6. Polarizing M: only difference is polarizer (polarizing filter)◦ Birefringence: ability of crystalline or Para -

crystalline material to rotate the phase of polarized light (double refraction) Skeletal muscle & Leydig cells *Amyloid protein: apple green ±Uric acid: negative ± ± Ca++ pyrophosphate

* ,±, ± ± clinical Significance ?

Types & Advantages of Microscopes

7. Electron M (EM): specimen is in vacuum◦Types: Transmission (TEM), scanning (SEM)◦Mechanism: similar to LM except that beam of

electrons replace light source◦Recording: photoelectric plate or video detector ◦Specimen preparation:

Fixation: Glutaraldehyde (cross links with proteins), Osmium tetroxide (reacts with *phospholipids) makes cell/tissue electron dense for image enhancement

Other steps are same as routine tissue processing except Plastic is used for embedding ± Diamond knives are used in microtome ( not metal knives)

◦ To study membranes – Freeze fracture technique {-160°C with glycerol (to prevent ice crystal formation)}

• * Where you find ?• ± why diamond knives are used in EM?

Types & Advantages of Microscopes

7. Scanning (SEM)◦ It differs from TEM that electron beam

passes across the surface of spectrum (not thru specimen as in TEM)

◦ Resembles Television◦ Can see 3D pictures

8. Atomic Force M: most powerful tool to study surface topography◦ Non – optical M: works like finger tip◦ Has highest resolution power – 50 pm◦ *Specimen need not be in vacuum

• *what is the additional advantage?