Tissue preparation

1. TISSUE PREPARATION
 Following specimens are used for IHC
1. Formalin Fixed Specimen
 The most popular choice of fixatives for routine histology are formalin based, usually a 10%
solution with the addition of phosphate buffers.
 Prompt fixation of thin (3 mm) slices of tissue is essential to achieve consistent
demonstration of tissue antigens.
 Delayed fixation or poor fixation may cause loss of antigenicity or diffusion of antigens into
the surrounding tissue.
 Following fixation most material is routinely processed to paraffin wax to facilitate section
cutting.

2. 2. Frozen section
 IHC on frozen sections remains an important histological tool.
 Frozen sections have certain inherent disadvantages compared to paraffin wax sections,
including poor morphology, limited retrospective studies and materials.
 The advantages of frozen sections is that the antigen is preserved and not-cross-linked or
hidden as can occur in paraffin wax sections.
 In practice, unfixed cryostat sections or cell preparations are used.
3. Cytological preparations
 The value of IHC on cytological preparations has been established.
 Acetone-fixed smears or cytospins are often preferred by the immunohistochemist, as this
allows a wide range of primary antibodies to be employed without destroying the target
epitopes.
 Where cytological samples are cellular enough, there is also the option of producing a cell
block which can be then be processed into paraffin wax.
 This gives greater flexibility for the number and range of tests which can then be
performed on the sample and also provides archive material.

3.  A prerequisite for all routine histological and cytological investigations is to ensure
preservation of tissue architecture and cell morphology by adequate and
appropriate fixation.
 Helps to prevent
 Elution
 Degradation
 Modification
 Preserves the position of the Ag
 Preserves the secondary and tertiary structure to a possible extent
 Provides target of Ab molecules
 Formaldehyde is the preferred fixative
 Most of the Ab available are optimized for use with formaldehyde
Fixation

4.  Paraffin wax has remained the most widely used embedding medium for diagnostic
histopathology in routine histological laboratories.
 Accordingly, the largest proportion of material for immunohistochemistry is formalin-fixed,
paraffin-embedded.
 Certain cell antigens do not survive routine fixation and paraffin embedding. So the use of
frozen sections still remains essential for the demonstration of many antigens.
 1-3 µm tissue sections are cut onto Poly L-lysine coated slide.
Embedding and
Sectioning

5. ANTIGEN RETRIEVAL
 When based fixatives are used, inter-molecular and intra-molecular cross-
linkages are formed with certain structural proteins.
 These are responsible for the masking of the tissue antigens.
 The degree of masking of the antigenic sites depends upon the length of time in
fixative, temperature and concentration of fixative.
 The demonstration of many antigens can be significantly improved by the
pretreatment with the antigen retrieval reagent that break the protein cross-
links formed by formalin fixation and thereby uncover hidden antigenic sites.
 Methods for antigen retrieval are:
i. Heat Induced Epitope Retrieval (HIER)
ii. Proteolytic Induced Epitope Retrieval (PIER)