immunohistochemical technique

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)

3. Historical aspect
Fixation for IHC
Antigen retrieval
Labels
Control

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,

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.

28. Mercuric chloride-containing fixatives are additive
and coagulative.
Suitable for the demonstration of intracytoplasmic
antigens.

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.

65. Enzyme labels
Colloidal metal labels
Fluorescent labels
Radiolabels

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.

70. Tissue/cells
• Need a good positive and negative
Reagent
• When trying new technique

71.  Indicate that proper staining technique was achieved
 Use the same control over time
• Follow staining consistency over time
• Familiarity with pattern

72. Maintenances
Stain first and last longer
Monitor antigenicity over time

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.