IMMUNOHISTOCHEMISTRY TECHNIQUES and SPECIAL PROCESSING

TECHNIQUE
Immunohistochemistry
 IHC includes in-situ detection of antigens in tissue sections and cells
using monoclonal and polyclonal antibodies
 Detection is through visualization of the antigen using microscope
 IMMUNO – Antigen/Antibody based
 HISTO – Tissue based
 CHEMISTRY – reaction
PROTOCOL FOR IHC STAINING 3. Peroxide Block
 Then rinse with Phosphate Buffered
1. Deparaffinization and Rehydration Saline (HK091-5K)
2. Antigen Retrieval 4. Power
3. Peroxide Block Block
4. Power Block
5. Primary Antibody
6. Super Enhancer
7. Secretory Antibody
8. Chromogen
9. Counter stain
10. Aqueous Mount
Protocol for IHC Staining
 Barriers slides are positively charged slides with
barriers to prevent loss of reagent. 5. Primary Antibody:
1. Dewaxing and Rehydration: Ez-  BioGenex has approximately 400
DeWaxTM Solutions antibodies, in concentrated and
 One-step Dewaxing and Rehydration reagent ready-to use formats.
 Free form xylene or xylene substitutes  Both Monoclonal and Polyclonal antibodies are available
 Then rinse with Phosphate
Buffered Saline (HK091-5K)

6. Super Enhancer:
2. Antigen Retrieval:  Enhances the reaction between
 Programmable time and temperature control the antigen and antibody
 Then rinse with Phosphate
 96 slides in one run
Buffered Saline (HK091-5K)
 Ease of use

 Phosphate Buffered Saline (HK091-5K) (rinse)
7. Secondary Antibody
 Polymer-HRP: Secondary
antibody conjugated with horse
9. Counter Stain
radish peroxidase enzyme.
 Hemotoxylin:
 Then rinse with Phosphate Buffered
Saline (HK091-5K)  Distilled Water
8. Chromogen:
 DAB: HK130 – 5K
 AEC: HK139 – 06K
 Then rinse with Phosphate Buffered
Saline (HK091-5K) Super Sensitive Link Label IHC Detection System
TM
10.
Aqueous
Super Mount
Sensitive TM HRP IHC Detection System
 Utilizes immunology + histo technology + chemistry
 Involves antibodies to antigens which are attached to color
reagent via a series of stages
 Study of site of antigen binding is demonstrated by direct
labelling of antibody or by means of a secondary labelling
method
 Aka Immunohistology
 Microscopic study of tissue with the aid of antibodies that bind
AUTOMATION IN IHC to tissue components and reveal presence
Immunoglobulins


Class of serum proteins produced by plasma cells and

lymphocytes
 IgA, IgD, IgE, IgG, IgM
 Protection against antigens, such as bacteria, viruses and toxins
 IgG  most commonly used in IHC
Epitope
 Structural part of the antigen that reacts with an antibody
(antigenic sites)
 Produce corresponding antibodies
 Group of amino acids in globular proteins & sugar -side chain
Definition of terms: polysaccharides

Immunohistochemistry Polyclonal antibodies Monoclonal antibodies

  Humoral antibody  Antibodies produced from a  fixation process can damage antigen that some antibodies will
 Produced by animal injected clone of plasma cells from a not recognize the antigen present in tissue
with purified specific molecule single cell immunized with  Tissue block can be stored for many years and archiving
that contains the antigen of specific immunogen
interest w/ various epitopes  Uniform homogenous Frozen Tissues (Cryostat section)
 Obtained via immunization with antibodies directed to specific  Snap-frozen in liquid nitrogen or isopentane
an antigen epitopes  Sections can then be cut using a cryostat which keeps tissue
 Collected from the Ig rich serum  Propagation is done by somatic frozen during processing
of rabbit, goat, pig, sheep, horse, cell infusion, selection of  Fixation is carried out immediately before staining using
guinea pig resulting hybridoma and absolute methanol or acetone in few seconds
limiting dilution o Fxn: preserve immunological activity and prevent
destruction of labile antigenic sites
 Disadvantage: Tissue morphology is generally not so good with
USES & APPLICATIONS OF IHC frozen sections
 Advantage: little antigen damage and most antibodies will react
1. ID of specific or highly selective cellular epitopes or antigens in as expected
frozen or paraffin-embedded tissues
2. Provides data about expression of antigens within the context of PRE-TREATMENT OF TISSUES FOR IHC/ ANTIGEN RETRIEVAL
tissue structure TECHNIQUES
3. Offers some important advantages over other techniques such as 1. PARAFFIN-EMBEDDED TISSUE
western blotting and flow cytometry a. Proteolytic digestion
4. Detect organisms in cytologic preparations such as in body fluids b. Heat Induced Epitope Retrieval (HIER)
sputum and FNAB 2. FROZEN TISSUE SECTION
5. Research and diagnostic labeling tool to differentiate malignant a. Peroxidase
tumors b. Alkaline Phosphatase

PREPARING TISSUE FOR IMMUNOHISTOCHEMISTRY PARAFFIN-EMBEDDED TISSUE

Two types of tissue processed in IHC
1. Paraffin-embedded tissue
2. Frozen tissue section
PARAFFIN-EMBEDDED TISSUE
 Also known as routine processed tissues
 tissues fixed with formaldehyde and embedded with paraffin
 best option to maintain cellular structure but have number of
issues in relation to IHC
 Tissues needs to be fixed in formalin immediately after
harvesting 4 to 24 hours
 5 to 10 microns section are produced by microtome prior to
labeling tissue sections are rehydrated
 Formalin fixation damage antigen due to cross-linking which
mean antibodies can no longer recognize antigen
 Sections are first deparaffinized, taken to alcohol and washed
a. Proteolytic digestion: done by incubation of tissue sections either
by Trypsin or Protease
 Advantage: demonstrates heavy chain IgG, complement and
specific antigens (Cytokeratin)
 Most common enzymes used:
1. Trypsin
- 0.1% Trypsin + 0.1% CaCl2 + distilled water
- Adjusted to pH 7.8 with sodium hydroxide preheated
at 37C and transferred to cold running water to stop
enzyme digestion
 2. Protease  Heated in a microwave oven for 10-60 mins (usually 20)
- 0.05 to 0.1% Protease + distilled water depending on the length of formalin exposure
- Adjusted to pH 7.8 with sodium hydroxide preheated
2. For Frozen Tissue
at 37C in distilled water and placed in protease
 Inhibition of endogenous enzyme that causes high background
solution for a shorter period of time
staining
b. Heat Induced Epitope Retrieval (HIER) 2a. Peroxidase
 May use either pressure cooker and microwave that have 3  Number of tissues containing endogenous peroxide which
cycles in heating at acidic buffer (pH 6) or alkaline buffer (pH 9) lead to high and non-specific background staining when
1. Microwave antigen retrieval using HRP system (Horse-radish peroxidase).
2. Pressure Cooking Antigen Retrieval  Blocking by pre-treatment with 0.3% Hydrogen peroxide in
3. Microwave and Trypsin Antigen Retrieval PBS (Phosphate Buffer Saline) or Methanol is recommended.
Microwave antigen retrieval
 Involves boiling of formalin-fixed deparaffinized sections in
solutions such as 0.01M citrate buffer
o EDTA at pH 8.0
o Tris EDTA at pH 9.9 or 10
 Time of boiling: 10 to 60 minutes (depends on the length of
formalin fixation)
 20 mins  most satisfactory time for most antigens and fixation
protocols
o Care should be taken not to allow the sections to dry after
heating = destroys antigenicity
o Adhesive – Vectabond coated slides in 10% formol saline 2b. Alkaline Phosphatase
for 1-2 minutes/ air dry before picking sections  Tissue containing endogenous AP can produce high
background when using an AP-conjugated antibody for
Pressure Cooking Antigen Retrieval
 detection.
 Less time consuming  Its activity is more prevalent in Frozen Tissue and can be
 Allows for more consistent recovery of many antigens, compared
 blocked by using pre-treatment with 5mM Levamisole.
to large batch microwave oven technique  FFPE (Formalin Fixed – Paraffin Embedded) tissues tend to
 Heat is uniformly distributed have much lower levels of endogenous enzyme activity so
 Slides are not subjected to hot spots & cold spots that results to blocking is not commonly required.
inconsistent antigen recovery
 Pressure @103 kPa or 15 psi in 120 C General Steps in IHC:

Microwave and Trypsin Antigen Retrieval A. For paraffin- embedded sections:

 Uses 0.1% trypsin in 0.1% CaCl2 in distilled water distilled water 1. Deparaffinization
adjusted to pH 7.8 with sodium hydroxide 2. Pre-treatment
3. Staining
 4. Coverslipping
5. Microscopic Evaluation
Deparaffinization
1. Deparaffinize sections in xylene 2 times for 5 minutes each tine
2. Hydrate with 100% ethanol 2 times for 3 minutes each time
3. Hydrate with 95% ethanol for 1 minute
4. Rinse in distilled water
5. Follow the procedure for pre-treatment as required
- if deparaffinization process in not complete: just repeat
the procedure
Pre-treatment
a) Proteolytic digestion
b) Heat Induced Epitope Retrieval (HIER)
For Frozen Sections:
Slide Preparation for Frozen Section
1. Snap-freeze fresh tissues in liquid nitrogen or isopentane
pre-cooled in liquid nitrogen, embedded in OCT compound
in cryomolds.
2. Store the frozen blocks at -80 Degree Celsius
3. Cut 4-8 m thick cryostat sections
4. Mount cryostat sections on either super-frost plus slides or
gelatin coated slides.
5. Store slides at -80 Degree Celsius
6. Prior to staining warm the slides at room temperature for 30
minutes and fix in ice cold acetone for 5 minutes. Air dry for
30 minutes
7. Wash in PBS (Phosphate Buffer Saline)
STAINING
 Principle In Histochemistry: Color product that is being product
from the reaction of some tissue the antigen bind to the Primary
antibody which is found in the reagent, and with the secondary
antibody it will form a complex with the aid of an enzyme that
will hazen (fastened) the reaction.
Procedure:
1. Rinse sections in PBS- Tween 2 times for 2 minutes each time
2. Serum Blocking: Incubate sections with normal serum block –
species same as the secondary antibody, for 30 minutes to block
nonspecific binding of immunoglobulin.
Note: This protocol uses avidin-biotin detection system.
Avidin-biotin block may be needed based on the tissue
type. Normal serum block should be used prior to the
avidin-biotin block.
3. Primary Antibody: Incubate sections with primary antibody at
appropriate dilution primarily antibody dilution buffer for 1 hour at
room temperature or overnight at 4 Degree Celsius.
4. Rinse in PBS-Tween 20
5. Peroxidase Blocking: Incubate sections in peroxidase blocking
solution for 10 minutes at room temperature
6. Rinse in PBS-Tween 20.
7. Secondary Antibody: Incubate sections with biotinylated secondary
antibody at appropriate dilution in PBS for 30 minutes at room
temperature
8. Rinse in PBS-Tween 20 for 3 times at 2 minutes each time.
9. Detection: Incubate sections in streptavidin-HRP in PBS for 30
minutes at room temperature
10. Rinse in TBS 3 times for 2 minutes each time.
11. Chromogen/Substrate: Incubate sections with DAB Solution for 1-3
minute
12. Rinse in PBS-Tween 20 for 2 times each time
13. Counterstain if desired
14. Rinse in distilled water
15. Dehydrate through 95% ethanol for 2 minutes, then 100% ethanol  (-) for non-epithelial tumors.
for 2 times for 3 minutes each time. b. CEA (Carcinoembryonic antigen) - found in carcinomas of
16. Clear in xylene the GIT, Pancreas, Lung, Breast, Ovary, Uterus, and Cervix to
17. Coverslip without mounting medium
differentiate Adenocarcinoma (CEA+) and Mesothelioma
(CEA-).
Principles in IHC
c. TTF1 (Thyroid Transcription Factor 1) - use to differentiate
i. Target Antigens or Tumor Markers lung adenocarcinomas from the mesotheliomas.
ii. Choosing Primary Antibodies  (+) in thyroid, lung and neuroendocrine tumors.
iii. Choosing Secondary Antibodies d. PSA (Prostate Specific Antigen)- used to diagnose prostatic
iv. Staining Protocols adenocarcinoma.
v. Microscopic Evaluation/Examination  Also (+) in certain pancreatic and salivary gland
tumors.
Target Antigens or Tumor Markers
B. Intermediate Filament Marker
1. Use for the diagnosis of tumors 1. Actin- sensitive marker for muscle differentiation and can be
2. Determination of tumor types used to identify tumors derived from smooth, skeletal and
3. Identification of infectious agents cardiac muscle.
4. Prognostic and therapeutic implications 2. Vimentin – 57 kD intermediate filament that is present in
normal mesenchymal cells and the plastic neoplastic
A. Epithelial Tumor Markers:
counterparts. Use to identify melanomas and schwannomas.
1. Keratin – highly sensitive marker for epithelial cells and present
3. Desmin- 53kD intermediate filament expressed by smooth and
in epithelial cells tumors or carcinoma includes:
striated muscle. Highly specific for myogenic tumors, including
a. CK7& (Cytokeratin 7) – found in carcinomas of the lung,
Leiomyoma and Rhabdomyosarcoma.
breast, uterus and ovaries.
4. Glial Fibrillary Acidic Protein (GFAP) – 51kD intermediate
b. CK20 (Cytokeratin 20) – common in carcinomas of the
filament protein expressed by CNS glial cells, particularly
colon and stomach.
Astrocytes. For diagnosis of Astrocytoma, Ependymomas, and
c. Both (+) for CK7 and CK20 – found in Transitional cell
carcinoma of the bladder and mucinous ovarian tumors. Medulloblastomas.
c. (-) for either CK7 and CK20 – found in Renal cell 5. Neurofilament (NF) – expressed in cells of neural origin,
carcinomas, thyroid carcinoma, hepatocellular carcinoma, particularly neurons, neurons processes, peripheral
prostatic adenocarcinomas, and squamous cell carcinomas. nerves, sympathetic ganglia and adrenal medulla.
6. S100 Protein – low molecular weight calcium binding protein in
CNS glial cells, Schwann cells, Melanocytes, Histiocytes and some
epithelial cells of the breast, salivary and sweat gland
2. Epithelial Tumor Markers: epithelium.
a. EMA (Epithelial membrane antigen) – high molecular C. Neuroendocrine Markers:
weight protein that is helpful in determining the size of the 1. Neuron-specific enolase (NSE) – iso enzyme marker that
tumor provides strong evidence of neural or neuroendocrine
 (+) in adenocarcinomas of the breast, lung kidney. differentiation.
2. Chromogranin – found in the neural secretory granules of the c. Melan-A (MART1) – encodes for the melanoma – specific
endocrine tissues and marker for neuroendocrine differentiation. antigen that is presented in normal pigmented cells of the
 (+) combination of keratin and chromogranin is skin and retina as well as in certain adrenocortical tumors.
typical in neuroendocrine carcinoma. G. Markers for Lymphomas:
 Chromogranin (+) and Keratin (-) may signify a. Leukocyte common antigen (LCA or CD45) – breast
paraganglioma. screening marker for lymphomas.
3. Synaptophysin- a 38kD transmembrane protein associated with b. CD3, CD4, CD8- is used for immunophenotypic
presynaptic vesicles of neurons. subclassification of lymphoma for T-cells.
c. CD19, CD20, CD23 – for subclassification of lymphomas for
D. Germ Cell Tumor Markers: B-cells.
1. Human Chorionic Gonadotropin (HCG) – synthesized by d. CD15, CD30 – for Reed Sternberg cells.
placental syncytiotrophoblasts and marker for choriocarcinoma. H. Cell Proliferation Markers:
2. Alpha-fetoprotein (AFP) – synthesized by normal liver cells and 1. Ki-67 (MIB-1) and Proliferating cell nuclear antigen (PCNA)-
marker for endodermal sinus tumors by showing yolk sac used to assess proliferation of tumor cells. Increased expression
differentiation. Also in embryonal carcinomas, teratomas and of these antigens is usually associated with greater
hepatocellular carcinomas. aggressiveness and higher likelihood of recurrence of metastasis.
3. Placenta-like alkaline phosphatase (PLAP) – produced by I. Cancer Associated genes:
placental syncytiotrophoblasts in the late pregnancy and used 1. p53 – a tumor suppressor genes that when mutated can
as a marker for germinomas and majority of seminomas. contribute to the development and progression of a malignant
phenotype of human tumors.
E. Mesenchymal Tumor Markers: 2. C-erbB-2, c-myc, ras- cellular oncogenes that can be used
1. Myogenic Tumor Markers (myo-D1,myoglobin and to diagnose breast cancer.
Myogenin) – Markers for tumors of skeletal muscle origin that J. Infectious Agent Marks
is (+) for muscle – specific actin and desmin. a. HAV
2. Fibriohistiocytic tumors (CD68, FAM 56) – when combined b. HBsAg
with the nonspecific proteolytic enzymes such as alpha-1- c. HBcAg
antitrypsin and alpha-1-antichymotrypsin is used to diagnose d. HVC
e. HPV
malignant fibrohistiocytic sarcomas.
3. Vascular Tumor Markers (Factor 7 related antigen, CD31 Associated with certain diseases or infection such as:
and Ulex Europaeus 1 (UEA) – endothelial markers for
1. Cytomegalovirus Epstein-Barr Virus
vascular tumors specifically angiosarcomas.
2. Toxoplasma
F. Markers for Melanomas:
3. Pneumocystis carinii
a. S100 Protein – reactive for melanocytes derived from neural
crest and usually the intensity of staining is inversely
4. Helicobacter pylori
proportional to the melanin content of the tumor.
5. Cryptosporidium
b. Melanosome (HMB-45) – widely used, high sensitive and
highly specific marker for the diagnosis of melanoma. 6. Cryptococcus neoformans
 7. Histoplasma
8. Entamoeba
9. Histolytica
10. Mycobacteria

Choosing Primary Antibodies

a. Specificity – specific in the epitope of the target antigen Choosing Secondary Antibodies
b. Exhibit minimal cross reactivity
c. Validated by clinical trials a. Specificity – specific in the isotope of the primary antibody
d. If using in FFPE, information on the requirements for antigen b. Can recognize the epitope of the primary antibody
retrieval is readily available c. Purification and absorption preparation
e. Commercial availability d. Can conjugate with enzymes and dyes and stains
f. Appropriate incubation conditions and working dilution e. Commercial availability and validated
f. Reactivity to human immunoglobulin classes, subclasses, types
and subtypes.

e. ABC Complex Technique:

(Avidin- Biotin complex)
Avidin- Derived from eggwhite
2. Direct Staining
a. Traditional direct Technique
b. Epos (Enhanced Polymer One Step Staining)

Staining Protocols:
1. Indirect Staining:
 utilizes labelled secondary antibodies making it more
specific and sensitive
Staining Techniques for IHC:
 Requires more time and Reagents
1. Indirect Staining  Readily available reagents and cheaper
a. Two –Step Indirect Technique 2. Direct Staining
b. Three - Step Indirect Technique  primary antibody is directly linked to either HRP or AP
c. Soluble Enzyme Immune Complement Technique (Unlabeled  Significant saving in time and reagents
Ab Tech)  No secondary antibodies required and fewer wash steps.
d. PAP (Peroxidase Antiperoxidase Technique)  simple dual staining techniques
 Limited availability of directly conjugated antibodies
and very expensive
 capable of bonding to the
previously bond secondary
reagent.
 Horseradish Peroxidase _ most commonly used enzyme for
indirect antibody enzyme – complex technique.

1.c Soluble Enzyme Immune Complex technique(Unlabeled Ab Tech)

 Utilizes perforemed
soluble enzyme – anti
enzyme immune complex
 Staining sequence
involves the use
of:
1a. Two-Step Indirect Technique a. Unconjugated
 Unconjugated primary primary Ab
antibody first binds to the Ag b. Secondary Ab
 An enzyme - labeled c. Soluble enzyme –
secondary antibody directed anti enzyme
against the primary antibody complex
(now the Ag) is then applied. d. Substrate solution
 Followed by substrate –
Chromogen solution
1d. PAP (Peroxidase Antiperoxidase Technique)
Note: If the primary antibody is made  Is an indirect Ab enzyme – complex
in rabbit or mouse, the secondary technique where the soluble PAP
antibody must be directed against complex is bound to unconjugated
rabbit or mouse IgG, respectively.
primary Ab (rabbit anti-human IgG)
 Cross reactivity is eliminated using pre absorb secondary
by a second layer “bridging” Ab and
Antiserum (antibody) absorbed with IgG.
the rabbit PAP complex.
 Horse radish peroxidase – enzyme
1b. Three- step indirect technique:
 DAB (Diaminobenzidine) – chromogen
 Second enzyme – conjugated Dark brown reaction – end
antibody is added to the previous product when Ag is present in the tissue.
method (two-step)
 Addition of the 3rd layer of the APAAP – Alkaline phosphatase – Anti – Alkaline Phosphatase Complexes:
antibody to further amplify the
*Advantage: Over that of the PAP – Lack of interference
signal, since more antibody’s are from endogenous peroxidase activity.
 Staining sequence:
 Recommended for the use on blood and marrow smear
 Endogenous alkaline phosphatase is usually blocked by adding a. Primary rabbit ( or mouse) Ab
Levamisole to the substrate solution. b. Biotinylated anti – rabbit (or anti
mouse) IgG and streptavidin enzyme
1e. ABC Complex Technique:
conjugate, the color reaction is then
 Avidin- Derived from eggwhite developed with the appropriate
substrate/ chromogen such as
Staining Technique: horseradish peroxidase.
a. Primary Ab
b. Biotinylated secondary Ab followed either by the preformed 1f. Labelled Streptavidin Avidin Biotin
( Strept) avidin – biotin enzyme complex of the ABC tech or by Technique
the enzyme labelled Steptavidin

Labelled Streptavidin Avidin Bio tech. (LSAB) 4 to 8 times more

sensitive than the old ABC method:
LSAB Procedure: 2a. Traditional Direct Technique
 Staining is sequence consist  Conjugate is primarily antibody
of primary rabbit ( or directly to the label, such as
mouse) antibody, fluorochrome, horseradish
biotinylated anti – rabbit peroxidase or AP
( or anti- mouse) Ig and  Advantage:
streptavidin – enzyme  It is a simple and quick
conjugate. The color since it requires by the
reaction is then developed appropriate chromogen
with the appropriate substrate solution.
substrate / chromogen, such as the horseradish peroxidase (HRP).  No secondary antibody
required thus fewer
wash steps are needed.
Single mouse on mouse
Direct Staining Procedure staining used.
 Disadvantage:
 Less sensitive.
 Cannot detect small amounts of Ag that could be
crucial in making the diagnosis.
  Limited and very expensive directly conjugated
 Large number of primary antibody molecules A/S and
antibodies.
peroxidase enzymes are attached to dextran polymer
2b. Epos (Enhanced Polymer One Step Staining) by DAKO “Backbone” or “Spine” molecule
 Advantage  Utilizes bright field microscope
 Rapid staining is  Easy to manipulate
completed in a single
step within 10 mins. In
addition to an average of
70 molecules of enzyme,
10 molecules of antibody
can be attached to the
spine molecule
 More sensitive than #1
and most suitable for
frozen section
immunohistochemistry
 Useful for both polyclonal and monoclonal antibodies
conjugation of both anti rabbit and anti- mouse
secondary antibody
 Avoid the use of Avidin (Strept) and Biotin
 Disadvantage
 Limited number of primary antibody commercially
available.

1. Light Microscopy:
 Employs visible light to study stained tissues

Microscopic Evaluation
2. Direct Immunoflourescense Technique for Solid Tissue Biopsies
 2-5 micra cryostat section of fresh unfixed material mounted
on the slide coated with gelatin adhesive or poly – L – Lysine
at 1:10 dilution
 Tissue is reacted directly with fluorescence – conjugated Ab.
Results of Px with lymphocytic lymphoma and macroglobulinemia:
 Apple Green fluorescene when fluorescein is used as
fluorochrome.
 Orange- red fluorescence with Rhodamine conjugates.

(39:35) Direct Immunoflourescense Technique for Solid Tissue

Biopsies
 Mainly used for detection of autoantibodies in the
patients serum, including anti-nuclear Ab (ANA)
antimicrobial Ab (AMA) and liver-kidney microsomal
Ab.

PRINCIPLES IN IHC:
V. MICROSCOPIC EVALUATION
2. Fluorescence Microscopy in Frozen Section
Immunocytochemistry
Used to identify antigens in fresh frozen sections
Fresh tissue should be frozen immediately & rapidly by
immersing directly in liquid nitrogen
Can use O.C.T. (Optimal Cutting Temperature Compound) as
supporting media to facilitate preparation of good quality frozen
sections
Requires several brief washing with Tris Buffered Saline (TBS) to
prevent contamination of reagents
Extending the driving period to 48 hours will result in improved Used to detect antibodies for the diagnosis of glomerular disease in frozen
morphology sections of renal biopsies
Applied to skin of patient with SLE &vasculitis to examine pattern of and the condenser system, which focuses the beam onto the object,
deposition of Immunoglobulins 2. The image-producing system, consisting of the objective lens,
Requires Fluorescence microscope movable specimen stage, and intermediate and projector
For examination of paraffin embedded tissue section lenses, which focus the electrons passing through the specimen
3. Electron Microscopy Types to form a real, highly magnified image
3. The image-recording system, which converts the electron
of Electron Microscopy
image into some form perceptible to the human eye. The
image-recording system usually consists of a fluorescent
screen for viewing and focusing the image and a digital camera
for permanent records.

2. Scanning Electron Microscope

The Scanning Electron

Microscope (SEM)uses a focused
beam of high energy electrons to
generate a variety of signals at the
surface of solid specimens. The signals
1. Transmission Electron Microscopy that derive from electron- sample
interactions reveal information about
 A microscope that uses accelerated the sample including external
electron as a source of illumination. morphology (texture), chemical
Because the wavelength of an composition, and crystalline
electron can be up to 100,000 shorter structure and orientation of
than that of visible light photons, the materials makingup the sample.
electron microscope has a high In most applications, data are
resolving power than a light collected over a selected area of the surface of the sample, and a 2-
microscope and can reveal the dimensional image is generated that displays spatial variations in these
structure of smaller objects. Can have properties.
magnifications up to about
10,000,000x.
 TEM has three essential systems:
HISTORY OFF EM
1. An electron gun, which produces
the electron beam,
Max Knoll & Ernst Ruska-
o invented the 1st electron microscope at Berlin
Technische Hochshule in 1931
Manfred Von Ardenne-
 o invented the earliest scanning electron microscope in lipids, is electron-dense, and stains phospholipids
1937 Ruska at Siemens, Germany produced the 1 st of thee cell membrane.
commercial electron microscope in the world
Radio Corporation of America’s (RCA) Model B
o Leader distributor in North America by Cecil Hall,
James Hillier & Albert Pretus of University of Toronto
Canada in 1983
Hitachi & Toshiba
o 1st in Asia to develop electron microscope in 1939
Sample Preparation for Electron Microscopy
1. Cryofixation
 Freezing a specimen rapidly, typically to liquid nitrogen
temperatures or below, that the water forms ice. This
preserves the specimen in a snapshot of its solution state
with the minimal of artefacts. Use to observe virtually any
biological specimen close to its native state.

2. Fixation
 Process of preserving a sample at a moment in time and
to prevent further deterioration so that it appears as close
as possible to what it would be like in the living state,
although it is now dead.. In chemical fixation for electron
microscopy, glutaraldehyde is often used to crosslink
protein molecules.
3. Rinsing
 Process of removing traces of aldehyde & osmium that
would contaminate the tissue.
 Uses sodium cacodylate buffer ph 5.1-7.4 to maintain the
appearance of the specimen.
4. Post-Fixation (Secondary Fixation)
 with osmium tetroxide (OsO4) increases the stability and
contrast of fine structure. OsO4 helps in the stabilization
of many proteins by transforming them into gels without
destroying the structural features. Tissue proteins
stabilized by OsO4 are not coagulated by alcohols during
dehydration. Osmium tetroxide reacts with unsaturated
5. Dehydration
 Removing water from the samples. The water is generally
replaced with organic solvents such as ethanol or acetone as a
stepping stone towards total drying for SEM specimens or
infiltration with resin and subsequent embedding for EM
specimens.
 30% Acetone or Ethanol=10 min
 50% Acetone or Ethanol= 20 min.
 70 Acetone or Ethanol= 20 min
 90% Acetone or Ethanol= 20 min
 100% Acetone= 3 x 20 min.
 100% Acetone = 20 min
6. Infiltration
 Epoxy resin is used to infiltrate the cells.
 It penetrates the cells and fills the space to give hard plastic
material which will tolerate the pressure of cutting. The epoxy
resin used for the 50:50 mixture can be from the frozen resin
stock. There are several epoxy resins (e.g. Spurr resin) have a
carcinogenic component and are useful for hard material like
following morning and polymerized further if necessary. A good test
for correct polymerization is to try and dent one of the side ridges of
the tip of the capsule with a fingernail the polymerization at 60
degrees Celsius should continue until the capsule is hard enough to
show no indentions. To remove the capsule from the mold, carefully
cut the mold lengthwise with a razor blade and peel the cut edges from
the top (not the tip) of the capsule. The capsule can then be easily
removed. If the side facets near the tip show cracks and/or bulging, it
usually indicates too rapid polymerization.
9. Trimming
 Excess plastics surrounding the tissue must be trimmed away
in a fashion that will yield a square or rectangular sections.
Trim the capsule while viewing under the dissecting
microscope using old glass knives or knives not suitable for
sectioning. The capsule mold must be trimmed to a pyramid
where the pyramid tip and sides are exposed tissue. The angle
of the pyramid sides (called facets) should be about 45
bone but should be used and disposed of with care. Since
most plastics dissolve in acetone and propylene oxide, the
samples must be dehydrated using ethanol and a series of
resin-ethanol mixes used during the infiltration process
(instead of the more usual resin-propylene oxide mixes).
Embedding is done using flat molds.
7. Embedding
 Infiltration of the tissue with a resin such araldite or LR
(London Resin) White, which can then be polymerized
into a hardened block for subsequent sectioning.
8. Polymerization
 In this step, tissues embedded in the resin (wrapped in
aluminum foil) are allowed to set overnight at room
temperature and then placed in an oven at 60 degrees
Celsius for 2-3 days. Specimens are placed in appropriate
molds, such as Beem capsules. Blocks may be sections the
degrees Celsius . Too steep of an angle will not allow
enough lateral support when sectioning while too flat (or
low) of an angle will cause the “face” being sectioned too
enlarge too quickly during sectioning. Use smooth slicing
(not chiseling) strokes that cut through the plastic in one
stroke. Take very thin slices so as to leave a smooth side
surface (important for good sectioning).
10. Sectioning
 The production of thin slices of the specimen. Must be
very thin so that they are semitransparent to electrons,
typically around 90 nm. These ultra-thin sections for
electron microscopy are cut on an ultramicrotome with
a glass or diamond knife. Glass knives can easily be
made in the laboratory and are much cheaper than
diamond, but they blunt very quickly and therefore
need replacing frequently.
11. Staining
 Uses heavy metals such as lead and uranium to scatter imaging
electrons and thus give contrast between different structures,
since many (especially biological) materials are nearly
“transparent” to the electron beam. By staining the samples
with heavy metals, we add electron density to it which results
in there being more interactions between the electrons in the
primary beam and those of the sample, which in turn provides
us with contrast in the resultant image.
 Positive stains
o Uranyl acetate
o Lead acetate
 Negative stains
o Ammonium molybdate
o Uranyl acetate
o Uranyl formate
o Phosphotungstic acid
o Osmium tetroxide
occur because of the accumulation of static electric fields due to the electron
irradiation required during imaging. It also increases the amount of
secondary electrons that can be detected from the surface of the sample in
the SEM and therefore increases the signal to noise ratio. Such coatings
include gold, gold/palladium, platinum, chromium etc.
DISADVANTAGES OF ELECTRON MICROSCOPY
 Electron microscopes are very expensive to buy and maintain.
 Requires stable high voltage supplies, extremely stable currents
to each electromagnetic coil/lens, continuously-umped
high/ultra-high vacuum systems and a cooling water supply
circulation through the lenses and pumps.
 Very sensitive to vibration and external magnetic fields,
microscopes aimed at achieving high resolutions must be housed
in buildings with special services.
 A significant amount of training is required in order to operate
an electron microscope successfully and electron microscopy is
considered a specialized skill.
 The samples have to be viewed in a vacuum, as the molecules
that make up air would scatter the electrons. This means that the
o Osmium ferricyanide
12. Freeze- fracture and freeze-etch-
 A preparation method particularly useful for examining
lipid membranes and their incorporated proteins in “face
on” view. The fresh tissue or cell suspension is frozen
rapidly (cryofixed), then fractured by simple breaking or
by using a microtome while maintained at liquid nitrogen
temperature. The cold, fracture surface is generally
“etched” by increasing the temperature to about -95 degrees Celsius
for a few minutes to let some surface ice sublime to reveal microscopic
details. Uses evaporated platinum and carbon, evaporated
perpendicular to the improve stability of the replica coating.
13. Spurr Coating
 An ultra-thin coating of electrically-conducting material,
deposited by low vacuum coating of the sample. This is
done to prevent charging of the specimen which would
samples need to be specially prepared by sometimes lengthy and
difficult techniques to withstand the environment inside an
electron microscope.
CHALLENGES IN IHC
1. No staining/ Weak staining:
Possible Things to Consider:
Primary antibody performance
o Check titer or dilution used, correct retrieval
methods Secondary Antibody Performance
o Optimize titer and check
specificity Consider antigen expression
o Does your tissue have this antigen?
Confirm substrate activity
Optimize incubation time and temperature Appropriate
use of (+) controls
Optimal tissue processing
o Poor fixation can damage
 2. High Background Staining  Highly specific secondary antibodies with no cross-
Possible Things to Consider: reactivity should be use
Are Endogenous enzymes & biotin blocked effectively? Primary 4. Mouse on Mouse Staining System:
antibody performance  Refers to using mouse models in studies and observations,
 Specificity as expected, Optimize titration specially mouse monoclonals for IHC staining
Secondary antibody cross Possible Things to Consider:
o Reactivity shows effective serum blocking and  Increase cross-reactivity of secondary antibodies with
titration endogenous mouse Ig
Optimize incubation time and temperature Increase  Use of weaker antibodies to mouse Ig
washing steps
Optimal tissue processing QUALITY ASSURANCE IN IHC:
o Poor fixation can cause background staining  Refers to a program in IHC that ensures reliability, accuracy and
3. Dual Staining precision of results.
 Refers to staining of antigens simultaneously to increase
antigen expression for studies and observation 3 Phases:
Possible Thigs to Consider: 1. Pre-Analytical – composes of pre-analytical procedures such as
 Has disadvantages if Indirect staining method was used  Tissue Identification
 Different primary antibody species or sub-classes are  Type
required  Dimension
 Laser resection  Accurate interpretation of results
 Thickness  Setting of (+) and (-) cut off levels
 Storage
 Drying 1. Internal Quality Control:
 Fixation method and Decalcification preparation  In staining quality control is very important
2. Analytica Phase – treatment procedures such as
 Primary antibody IHC controls:
 Its dilution  Test for specificity of the antibodies involved
 Buffer  Avoid misinterpretations due to false (+) or false (-) results
 Time and temperature requirements
Staining methods ( Control – section of known and proven antigen in question
 Manual or automation (-) Control – done using a parallel section from the tissue by omitting
 Development the primary antibody or replacing the specific primary antibody by an Ig
 Sensitivity and specificity that is directed against an unrelated antigen.
3. Post-Analytical Phase
 Use of controls Internal Tissue Control:
 Qualification and proper selection of controls
 Reporting of results
 a.k.a “built-in” control, this eliminates the variable of tissue fixation  but cannot identify a poorly calibrated IHC system giving
between specimens and controls but it contains the target antigen, insufficient staining results
not only in the tissue elements under investigation  EXAMPLES:
 Goat serum (normal)
 Mouse IgG
 Mouse serum (normal)
 Rabbit Ig Fraction (normal)
 Rabbit serum (normal)
 Swine serum (normal)
 Universal (-) control mouse
 Universal (-) control rabbit

2. External Quality Assurance:

 Standardization of IHC staining methods are generally
not possible
 Standardization of IHC staining reactions and
interpretation is vital for reliable and comparable results
 Process of agreeing upon technical standard based on
guidance documents to the state and develop through
expert consensus
 Staining quality varies greatly between different labs
depending on the individual selection of methods and
technical expertise
 Quality of commercial antibodies, reagents and
guidelines are varying
 External QC and Proficiency testing should be mandatory
 Construction of the organization or agency that will cater
EQAS
 Provides objective evidence of Lab proficiency
 Identifies methodological errors
 Provides directions for improvements

NordiQC:
 Accredited agency that is found in the Institute of Pathology,
Aalborg Hospital, Denmark.
 Caters laboratory across UK, Canada, Australia, and US
 No available EQAS provider in the country
 www.nordicQC.org
In-Situ Hybridization:
 Type of hybridization that uses a labelled complementary DNA,
RNA or modified nucleic acids strand to localize a specific DNA
or RNA sequence in a portion or section of tissue (in situ), or, if
the tissue is small enough, in the entire tissue, and in circulating
tumor cells (CTCs)
 Based on the specificity of the interaction of a probe with the
target nucleic acid, rather than the target protein or immunogen
 Uses Formamide and Heat as denaturants to separates nucleic
acid of double-stranded helix into 2 single strands
 In hybridization assay, the 2 sources are the target (sample) and
the probe are nucleic acids
 Probe is known fragments of nucleic acid with label that can be
detected. Produced by either recombinant nucleic acid
technology or through chemical synthesis
 Utilizes hybridization reaction where the sample searched for
specific nucleic acid sequences

Estrogen and Progesterone Receptor Assay (ER/PR Assay) from

Paraffin Section

I.
 Immunochemistry – important tool in diagnostic
pathology
 Large selection of antibodies is useful as markers for cell
function, cell type, or cell differentiation
Introduction
 Treatment of proteolytic enzymes under carefully
controlled conditions may demask epitopes, making
 proteolytic enzymes digestion as a prerequisite for
obtaining optimal immunostaining
 Introduction of the microwave oven
immunohistopathology has opened a new world of
possibilities
 Simple treatment of tissue sections in the microwave oven
may for some antibodies replace the enzymatic digestion
 Determination of steroid hormone receptors has become
widely used in the management of hormone dependent
cancers
 Estrogens receptor content of breast carcinoma is a
predictor of both prognosis and response to endocrine
therapy
 Progesterone receptor is an estrogen regulated protein
 Presence of progesterone receptor in human breast cancer
has been proposed as a mechanism whereby tumor cells
responds to estrogen
II. Fixation
 10% Neutral buffered Formalin
III. Reagents (Contents on the Kit)
1. Blocking Reagent
2. Primary antibody (Lyophilised)
3. Secondary antibody
4. Reagent A: Avidin
5. Reagent B: Biotinylated horseradish peroxidase
6. Hydrogen peroxidase solution
7. Diaminobenzidine tablets
IV. Preparation of Reagents:
1. Lyophilised Primary Antibody:
 Reconstitute 1 vial using 2 ml distilled water
2. Secondary Antibody:
NOTE: DO NOT PLACE CLOSE TOGETHER, UNEVEN STAINING MAY
OCCUR
 Secondary Ab …………………………… 1ul
 Tris buffered saline (TBS) …………. 499 ul
in 3. ABC Reagent:
 TBS…………………………………………... 100 ul
 Reagent A ………………………………… 1 ul
 Reagent B ………………………………… 1 ul
4. Hydrogen Peroxide Solution:
 HO 2 2 …………………………………………. 200 ul
 Distilled water ………………………….. 5800 ul
5. DAB Tablet Solution:
 To 10 ml TBS add 1 DAB tablet using plastic forceps
 Once completely dissolve add diluted hydrogen
peroxide solution – 0.2 ml
 Mix well and filter to remove any particles
6. Tris Buffered Saline (TBS):
 Dissolve 60.67 gm Tris base in 500 ml distilled
water
 pH to 7.6 with 1 N HCl to 1000 with distilled water
7. 0.01 M Sodium Citrate buffer (pH 6.0)
 Sodium citrate ……………………………….. 2.94 gm
 Distilled water ……………………………….. 1000 ml
pH to 6.0 with 1M NaOH
V. Staining Protocol:
1. Cut and mount sections on slides coated with vectabond
reagent
2. For better adhesion of tissue sections incubate slides (with
tissue) for 24 hours at 56oC oven
NOTE: TEMPERATURE SHOULD NOT EXCEED 60oC
3. Deparaffinized sections and rehydrate to distilled water
4. Wash with sodium citrate buffer for 2 changes, 20 dips each
5. Prepare primary antibody
6. Positions slides into glass staining racks and place in a 1000
ml beaker. Add sodium citrate buffer, the resulting volume
should be 800 ml
7. Position baker inside the microwave and heated for 2, 5
mins cycles at full power
 8. Let the sections stand for 20 mins in the microwave.
(THIS IS VERY IMPORTANT) REPORTING WOULD BE: POSITIVE OR NEGATIVE
9. Wash sections in TBS for 2x5 mins
10. Dry slide around edge of section using paper tissue and STAINING INTENSITY:
immediately cover the section with blocking reagent for 0 = No labelling
10 min in a humidified chamber + = Weak labelling
11. Decant excess serum (DO NOT WASH) + = Intense labelling
12. Cover section with primary antibody and incubate for 60 + = Very intense labelling
mins at 25oC in a humidified chamber
13. Wash in TBS for 2x5 mins PERCENT OF TUMOR CELLS STAINED:
14. Apply secondary antibody (100 ul Per section) and 0 = 0 to 10% Tumor cells stained
incubate for 30 mins at 25oC + = 11 to 25% Tumor cells stained
15. Prepare ABC reagent + = 26 to 50 % Tumor cells stained
16. Wash TBS for 2x5 mins + = 51 to 75% Tumor cells stained
17. Cover section with ABC reagent (100 ul Per section) and + = More than 75% Tumor cells stained
incubate for 30 mins at 25oC
18. Prepare DAB tablet solution
IMMUNOHISTOCHEMISTRY STAINING USING THE HISTOSTAIN – SP
CRITICAL STEPS AND TIPS TO CONSIDER KIT
1. Adhesion of tissue sections
2. Buffer for microwave treatment STAINING PROTOCOL:
3. Post treatment incubation
4. Primary antibody SEPECIMEN PREPARATION:
5. Secondary antibody  Survival of tissue antigen for immunochemical staining may
6. Preparation for ABC reagent depend on the type and concentration of fixative
7. Prepare DAB solution right after application of ABC reagent  10% neutral phosphate buffered formalin is generally
8. Be sure to dry slide around the edge of section using tissue paper recommended as the best fixative
of gauze. DO NOT TOUCH THE SECTION
9. Do not allow section to dry out B. PARAFFIN EMBEDDED TISSUE:
10. It is important that the immunohistochemical labelling NOTE: Temperature during tissue processing must not exceed 60oC,
procedure is carried out at a temperature of 25 oC and in a because rapid high temperature destroys antigenicity
humidified chamber
A.1. SLIDE PREPARATION
SCORING SYSTEM: 1. Precoat slides with histogrip or 0.1% L-Lysine in water, then air
dry
2. Cut paraffin sections at 3 microns
A.2. REMOVAL OF PARAFFIN AND DEHYDRATION OF TISSU