* Treponema pallidum causes syphilis. * A highly aneuploid tumor indicates genomic instability which is associated with increased aggressiveness and poor prognosis. * Amyloid protein showing apple green birefringence under polarized light microscopy has clinical significance as it indicates amyloidosis. * The signs ±, ± ± indicate the type of birefringence shown by substances under polarized light microscopy. ± indicates negative birefringence shown by uric acid while ± ± indicates positive birefringence shown by calcium pyrophosphate. * Osmium tetroxide reacts with phospholipids in cell membranes, making them electron dense and visible under electron microscopy. This helps in better structural visualization of cells

1. Methods to study Histology
Krishna T

2. Histology
 Cell
 Tissue
 Organ
 Organ system
 Homeostasis

3. How to get the histology slides?
 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

4. 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

5. Special situations
 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

6. Basis of staining
ACIDIC DYES BASIC DYES
Eosin Hematoxylin /Methylene
blue
Carry net negative charge Carry net positive charge
React/bind with cationic With anionic components of
components of the cell/tissue
cell/tissue
Less specific (as compared Highly pH specific
with basic dyes)
Acidophilic / Eosinophilic Basophilic substances
(cytoplasmic filaments, ( Po4 of Nucleic acids, So4
intracellular membranous of MPS, CO proteins)
components, extracellular
fibers)

7. What is special about Hematoxylin?
 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

8. Metachomasia
 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

9. PAS =Periodic Acid Schiff
 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

10. Feulgen stain for Nuclear Proteins
 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)
Why RNA cannot be stained by Feulgen?

11. Enzymatic digestion
 For the confirmation of specific substances
 Pretreatment of sections with specific enzymes
 Diastase/amylase  for glycogen
 DNA ase  for DNA

12. Enzyme Histochemistry
 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
enzyme
 AB (substrate) + T (trap) AT ( reaction
product) + B (Hydrolyzed component of substrate)

13. Immuno Histo Chemistry (IHC)
 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

14. Monoclonal Antibodies
Specific antigen Multiple Myeloma pts.
(actin of rat)
↓
B lymphocytes of
Immunized rabbit
Monoclonal B ells
Hybridoma cells
↓
Single specific type of antibodies
(Monoclonal)
( against Actin)

15. Clinical Significance of Monoclonal
Antibodies
 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

16. Immunological Methods
 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

17. 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

18. 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

19. orientation of cut
 Click to edit Master text styles
◦ Second level
◦ Third level
 Fourth level
 Fifth level

20. Three dimensional picture
 Click to edit Master text styles
◦ Second level
◦ Third level
 Fourth level
 Fifth level
How you get it?

21. 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?

22. 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?

23. 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 ?

24. 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 ?

25. 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?

26. 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?