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IHC
1. 1
Presented by
Dr. Shrikant Sonune
Guided by
Dr Ashok Patil,
Dr Shilpa Kandalgaonkar,
Dr Mayur Chaudhary,
Dr Suyog Tupsakhare,
Dr Mahesh Gabhane.
Immunohistochemical
technique (part II)
4. • 1941: Coons et all. have demonsrated antigens on tissue
sections by using an antibody which was linked to an florescent
label.
• 1966: Nakane and Pierce as well as Avrameas and Uriel
reported the use of secondary antibody which was conjugated
with peroxidase enzyme.
• 1970 : Sternberger et al described the peroxidase-
antiperoxidase technique.
• 1971: Envall & Perlman reported the use of alkaline
phosphatase labeling
5. • 1975: Kohler & Milstein described a revolutionary
method for production of monoclonal antibodies
• 1976 : Huang et al described the use of trypsin
digestion on paraffin sections as a means of reveling
some antigens otherwise masked by formalin fixation &
paraffin processing.
• 1977:Heggness & Ash proposed the use of avidin –
biotin for immunofluorescence.
6. • 1979: Sternberger described the peroxidase-antiperoxidase
method.
• 1981: Hsu et all. have defined the method of avidin-biotin-
peroxidase complex which is abbreviated as ABC method.
• 1984 : Alkaline phosphatase anti alkaline phosphatase
technique using a red reaction product.
• 1993: Advent of one step system, based on a two layer
dextran polymer. Reported by Pluzek et al at UK pathological
society meeting.
7. 1994 : Norton reported that the stainless steel
pressure cooker was more efficient than the
microwave oven for retrieving some antigens & this
was conformed by Miller et al.
1998: Dako launched a new labeling system based
on the dextran polymer technology.
8. • Intercellular antigens,
• Cell surface antigens,
• Tissue antigen for
diagnosing autoimmune
diseases
• Protein hormones in
histopathological diagnosis
• Soluble antigens of the cell
• Diagnosis of the endocrine
tumors
• Small amounts of peptides
in endocrine or
neuroendocrine cells
• Immunodeposits
• Tumoral markers
• Tumor typing
9. 1. Fixation
Fresh unfixed, fixed, or formalin fixation and
paraffin embedding
2. Sectioning
Tissue
preparation
10. 1. Antigen retrieval
Proteolytic enzyme method and Heat-induced method
2. Inhibition of endogenous tissue components
3% H2O2
Pretreatment
11. Make a selection based on the type of specimen,
the primary antibody, the degree of sensitivity and
the processing time required.
Staining
12. The requirements of a fixative vary according to the
different techniques employed
13. Blood smears are preserved by air-drying.
The subsequent fixation method used depends upon the
staining technique.
Routine air-drying for 1–2 hours does not have a
deleterious effect on most antigens studied
immunocytochemically.
Routine air-drying for 1–2 hours does not have a
deleterious effect on most antigens studied
immunocytochemically.
14. Most cytology smears are immediately fixed in 95%
ethanol or are spray-fixed with a carbowax
containing alcoholic fluid.
Ethanol fixation prevents staining for most leukocyte
markers, such as T and B cell antigens.
15. Two preparations can be made,
one wet-fixed
one air-dried.
With wet-fixed smears, one of the main problems is
the loss of cells, particularly clumps, during the
Immunostaning incubations.
16. Cryostat sections give much better antigen
preservation than paraffin sections.
Additionally, fixative can be used with cryostat
sections,
Different and optimal fixative for each antigen,
17.
18. The most popular fixatives contain
Formalin (40% w/v formaldehyde in water),
A neutral salt to maintain tonicity
A buffering system to maintain pH.
Well tolerated by tissues
Have good penetration.
Generally formalin-based fixatives are excellent for
most Immunostaning procedure.
19. Formaldehyde fixes tissue by reacting primarily with
basic amino acids to form cross-linking “methylene
bridges.”
Relatively low permeability to macromolecules and that
the structures of intracytoplasmic proteins are not
significantly altered.
The great variation in time and conditions for fixation
that cause majority of problems in immunochemistry.
20. Many antigens can be demonstrated after the use of
appropriate pretreatment methods,
Such as proteolytic enzyme digestion and/or antigen
retrieval, particularly if polyclonal antisera are used.
21. If monoclonal antibodies are to be utilized on
formalin-fixed, paraffin-embedded tissue sections,
then
Does formaldehyde react with the epitope under
investigation?
Does it react with adjacent amino acids causing
conformational changes?
Does paraffin processing destroy the epitope under
investigation?
22. If there are conformational changes resulting from
the reaction of formaldehyde with amino acids
adjacent to the epitope,
These can often be reversed using proteolytic
enzyme digestion or antigen retrieval.
23. If there are conformational changes in the epitope
due to tissue processing, these are irreversible.
Many epitopes are sensitive to heat, and during the
paraffin-embedding step, tissues are heated to the
melting point of wax, usually between 50–60°C.
24. Saturated (1.2% w/v) aqueous picric acid 75 mL
Formalin (40% w/v formaldehyde) 25 mL
Glacial acetic acid 5 mL
A saturated solution (w/v) contains 1.17 g/100 mL
distilled water.
25. This fixative penetrates rapidly and fixes all tissues very
well,
Fixation time is 1–12 hours
Lipid-containing antigens may be affected.
Tissues fixed in Bouin’s solution must be washed in
70% ethanol to precipitate soluble picrates prior to
aqueous washes.
After cutting sections, the yellow color in the tissue can
be removed by treatment with 5% (w/v) sodium
thiosulfate, followed by a water wash.
26. To improve cytological preservation and minimize
distortion associated with formaldehyde-based
fixatives, mercuric chloride-based fixatives are used.
These fluids are generally poor penetrators and are
not well tolerated by the tissues.
27. Frequently, mercuric chloride-based fixatives are
used secondarily to formalin.
Tissues are initially fixed in formal saline or neutral
buffered formalin.
Tissues are then immersed in the mercuric chloride-
containing fluid for a further period of fixation.
29. The permeability of the tissue is greater
Coagulant fixatives,
Antibody penetration is better,
Resulting in a more intense Immunostaning.
These fixatives are also having additive effect.
30. B5 is widely advocated for the fixation of lymph node
Biopsies
B5 STOCK SOLUTION
Mercuric chloride 2 g
Sodium acetate 2.5 g
Distilled water 200 mL
B5 WORKING SOLUTION
B5 Stock solution 20 mL
Formalin (40% w/v formaldehyde) 2 mL
31. ZENKER’S STOCK
SOLUTION
Mercuric chloride 50 g
Potassium dichromate 25 g
Sodium sulfate 10 g
Distilled water to 1 liter
ZENKER’S WORKING
SOLUTION
Zenker’s stock solution 100
mL
Glacial acetic acid 5 mL
CLEARING SOLUTION A
Iodine 0.5 g
70% ethanol 100 mL
CLEARING SOLUTION B
Sodium thiosulfate 5% (w/v)
in distilled water
32. Morphological detail is generally well preserved,
Penetration is poor
Fixation time is 2–15 hours at room temperature,
Sections are washed in running water for at least 1
hour to remove potassium dichromate deposits.
33. The tissue is then dehydrated using clearing agent A,
helps to remove mercuric deposits prior to sectioning.
After cutting sections and collecting on clean glass
slides,
The tissue again incubated in clearing solution A for 1–2
minutes.
Sections are rinsed in water, and placed in clearing
solution for 1–2 minutes.
34. This fixative is becoming increasingly popular, partly
because it preserves membrane proteins.
Zinc chloride 500 g
Formalin (40% w/v formaldehyde) 3 L
Glacial acetic acid 19 mL
Distilled water 20 L
35. WORKING SOLUTION
3% (w/v) Paraformaldehyde 50 mL M
Disodium hydrogen orthophosphate 100 mL
Lysine 0.9 g
Sodium periodate 0.15 g
Adjust to pH 7.4.
36. First described by McLean and Nakane
Preserve the micro anatomical relationship of cells
and cytological details.
Since the periodate oxidizes sugars to produce
aldehydes which are cross linked with lysine.
The paraformaldehyde stabilizes proteins.
37. A recent modification to this solution involves the
addition of potassium dichromate (PLDP).
This chemical is added to preserve lipids and the
fixative should therefore preserve all protein,
carbohydrate and lipid antigenic determinants.
38. It must be noted, however, that since additive
compounds are formed, immunoreactivity may be
blocked.
Furthermore, cytological detail is not as good as
with other fluids.
39. Sainte-Marie first described the use of alcohol
fixation and paraffin processing for
immunocytochemistry.
Small pieces of tissue are fixed rapidly and show
good cytological preservation.
40. They are in some ways ideal fixatives for
immunocytochemistry.
Since alcohols are coagulant fluids and do not form
additive compounds,
They permit good antibody penetration and do not
block immunoreactive determinants.
41. Conformational changes can occur.
Alcohols precipitate carbohydrates
Hence, useful for surface membrane antigens,
which often display carbohydrate-containing
epitopes.
42. Applied to frozen sections or smears.
Proteolytic digestion or antigen retrieval is of no use
following alcohol fixation
Ethanol fixation results in destruction of the tissue
section or smear.
43. Acetone is an excellent preservative of immunoreactive
sites, leaving most sites intact.
But it is a very poor penetrator.
It is used only for smears and cryostat sections.
Fixation is not complete, and extended incubation in
buffers may result in chromatolysis and loss of
membranes.
44. The dilemma of trying to obtain good morphological
preservation while maintaining immunoreactivity is
even more apparent in ultra structural
immunochemical studies.
For routine electron microscopy, it is usual to
employ glutaraldehyde primary fixation followed by
post fixation in osmium tetra oxide.
45. This combination produces excellent ultra structural
detail with good preservation of membranes.
With immunochemistry, however, the combination of
glutaraldehyde and osmium tetra oxide is not
generally useful.
46. Pre treatment of ultrathin sections with hydrogen
peroxide or sodium metaperiodate to counteract the
deleterious effects of osmium.
The glutaraldehyde primary fixation is not suitable
for many antigens, at least when employing the
concentrations of glutaraldehyde used for routine
electron microscopy.
47. WORKING SOLUTION
0.2 M Phosphate buffer, pH 7.4 50 mL
8% (w/v) Paraformaldehyde 25 mL
25% (w/v) Glutaraldehyde 0.8 mL
Distilled water 24.2 mL
48. If the tissue is frozen
The sections may need to be used in
immunohistochemistry
Acetone fixed:
- precipitates proteins onto cell surface---may extract lipids
- is needed for many of the “CD” antibodies
Unfixed:
Advantage: antigens are unaltered
Disadvantage: sections may fall off slide during staining
Paraformaldehyde fixed:
- needs to be freshly made, or frozen soon after
49. - Deparaffinize ( remove the infiltrated paraffin wax, by using organic solvents)
- The section then needs to be rehydrated, by sequential immersion
in graded alcohols (100%, 70% , 50% and then PBS)
- The deparaffinized section may need to be treated to expose buried
antigenic epitopes with either proteases or by heating in low pH citrate
buffer , or high pH EDTA buffer (Antigen Retrieval)
If the tissue is paraffin embedded
50. The loss of immunoreactivity by many antigens
caused as a result of fixation in formalin.
Hence many challenges for doing IHC in such cases
51. Epitopes 2 types
Formalin resistant- even after routine formalin fixation
this type of epitopes remains unchanged
Formalin sensitive- after routine formalin fixation this
type of epitopes shows substantial changes. It can be
divided into
• Irreversible
• Reversible or “Masked”
Rescued by antigen retrieval
52. The first attempt to “improve” the immunoreactivity
of formalin-fixed tissue antigens was by use of
tryptic digestion prior to immunofluorescent staining.
Proteolytic digestion compensates for the
impermeable nature of cross linked fixatives and
allowing hidden determinants to be exposed.
53. The concept of recovering lost immunoreactivity
Through exposure to heat near the boiling point of
water
But ,went against the tenet of protecting proteins from
the denaturing effect of heat.
54. The principle of antigen retrieval relies on the
application of heat for varying lengths of time to
formalin fixed paraffin embedded (FFPE) tissue
sections in an aqueous medium.
After deparaffinizing and rehydrating the tissue
sections, the slides are immersed in an aqueous
solution commonly referred to as a “retrieval
solution”.
55. The following techniques can retrieve many masked
antigens in routinely processed tissue:
Proteolytic enzyme digestion
Microwave antigen retrieval
Microwave and trypsin antigen retrieval
Pressure cooker antigen retrieval.
56. Pre- treating formalin-fixed routinely processed
paraffin sections with proteolytic enzymes to
unmask certain antigenic determinants was
described by Huange et al. (1976), Curran &
Gregory (1977). and Mepham et al. (1979).
The most popular enzymes-Trypsin
-Protease
57. It is generally accepted that the digestion somehow breaks
down formalin cross linking.
Hence the antigenic sites for number of antibodies are
uncovered.
Pure trypsin has a very limited digestion effect on formalin
fixed paraffin section
Essential for the demonstration of Cytokeratin & CD3.
58. Pitfalls of proteolytic digestion .
Under digestion results in very little staining.
Over digestion can result in very sever tissue
damage.
Batches of trypsin varies even from same source.
59. Pitfalls of proteolytic digestion .
Many antigens do not retrieved .
Some even destroyed by proteolytic enzyme.
The specificity of some primary antigens altered.
Allergy to trypsin
60. Remains largely unknown
Heat is obviously of great importance in reversing the
damages caused by the fixation with formalin and
embedding in paraffin.
Some cross-links are reversible (Schiff bases), thus
restoring the immunochemical integrity of the protein,
while others are not (methylene bridges).
Heat-induced AR has been more successful than the
use of proteolytic enzymes
61. Microwave oven heating retrieves many antigens
though previously lost or destroyed by routine
histopathological method.
Dewaxed section of tissue is boiled with the various
solutions like m-citrate buffer pH 6.
The prerequisite for this technique is use of strong
adhesive such as Vectabond or amino
propyltriethoxysilane(APES).
62. Replacing microwave oven with pressure cooker.
Optimized microwave oven heating not produce
consistent result.
Comparatively this method is consistent & less time
consuming.
Also require the strong adhesive.
63. Reported by Sandison et al in 1994
Heat pretreatment increases the sensitivity of
sections to the subsequent proteolytic digestion.
Drastically reduces the trypsin time
64. Advantages
Dilution factor of primary antibodies are greatly
increase.
Labeling of other antigens
Disadvantages
Proteolytic enzymes can destroy the sections.
Staining distribution can be altered.
66. Enzymes are most widely used labels.
Chromogen using standard histochemical method
produce a stable colored reaction end product
suitable for light microscopy.
Horseradish peroxidase is most widely used
enzyme.
67. There are also several chromogens for demonstration
of peroxidase.
Diaminobenzedine(DAB)tertrahydrochloride- brown
3-amino-9-ethylcarbazole- red
4chloro-l-naphthol-blue
Hanker-Yates reagent-dark blue
Alpha-naphthol pyronin –redish purple.
68. Colloidal gold is most common metal tracer in use.
Pink in color when observed in transmitted light.
Silver participation reaction can be used to amplify
the visibility of gold conjugate thereby allowing the
use of smaller, more stable gold conjugate.
Colloidal gold has much wider usage with the
electron microscope.
71. Indicate that proper staining technique was achieved
Use the same control over time
• Follow staining consistency over time
• Familiarity with pattern
73. Internal control
• Built-in or intrinsic controls
• Target antigen within normal tissue elements in addition
to tissue elements being evaluated
• Can replace external positive control
74. Internal control examples:
• S-100 protein in both melanoma and normal tissue
• Desmin present in blood vessel musculature
75. Used to determine antibody specificity
Use tissue without antigen expression
May need to identify negative cells within tissue
76. The ideal negative control is omission of primary
antibody.
In absorption control the primary antibodies are used
but not in pure form.
Pre-absorption of primary antibody with purified antigen.
This type of control is used in characterization &
evaluation of new antibodies.
77. The bonding between the primary & secondary
antibody is prevented in indirect technique by use of
some control.
This type of control is know as blocking control.
78. Textbook of microbiology Anathnarayan.6th
edition
Theory & practices of histologycal
thecnique – bancroff 5th edition
Immunohistochemical Staining Methods
Fourth Edition – Dako.
Science of lab diagnosis by john croker .
2nd edition.