Methods to-study-histology
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Transcript of Methods to-study-histology
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?