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Krishna T
Histology
Cell
Tissue
Organ
Organ system
Homeostasis
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
How to get the histology slides?
6. section cutting 5-10 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
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
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
Feulgen stain for Nuclear Proteins
enzymes
Diastase/amylase for glycogen
DNA ase for DNA
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
enzyme
ases
AB (substrate) + T (trap) AT ( reaction
product) + B (Hydrolyzed component of
substrate)
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 Rats actin/ anti rat actin
antibodies) bind with Fluorescent dye
Monoclonal Antibodies
B lymphocytes of
Immunized rabbit
Monoclonal B ells
Hybridoma cells
Single specific type of antibodies
(Monoclonal)
( against Actin)
Clinical Significance of Monoclonal
Antibodies
lymphomas)
Treatment Anti-TNF- antibodies in
inflammatory disorders
Immunological Methods
Immuno -fluorescence
Direct (one step, less sensitive)
&
Indirect ( more sensitive, Expensive, labor intensive,
cant easily run in automated) methods
Immunoperoxidase method
Enzyme is used ( horse raddish peroxidase) to color
colorless substrate into colored insoluble product
Other Methods
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
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 (cant 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
* ,, clinical Significance ?
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