Methods to study histology

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?