This document provides an overview of an advanced histopathology techniques course. The course covers topics related to immunohistochemistry (IHC), immunofourescent methods, enzyme histochemistry, and in-situ hybridization. IHC is described as the localization of antigens in tissue sections using labeled antibodies and visualized with markers. Key steps in IHC include sample preparation, antigen retrieval, blocking background staining, antibody incubation and detection, and counterstaining. IHC is useful as a diagnostic tool, for assessing disease progression and cancer treatment, and differentiating cell types.

1. Advanced Histopathological
Techniques

2. Histological techniques
of preparation

3. Histopathology & cytology
 Histopathology: microscopic examination of tissue
(cells) in order to study abnormality as a result of
disease

4. Advanced Histopathological
Techniques

5. Prerequisites
 Cell biology- Normal histology
 Histopathological techniques
 Pathology
 Cytology

6. Topics:
 1: Introduction (course out lines, concepts in IHC)
 2: definitions, Production of antibodies, sample preparation, antigen retrieval
 3: Antibody labeling, IHC methods (1),
 4: IHC methods (2), detection of low level antigens , controls in immunohistochemical
staining,
 5: Applications of IHC , tumor markers (intermediate filaments)
 6: tumor markers (Epithelial related antibodies,Germ cell tumor marker,
Neuroendocrine marker )
 7: tumor markers (hormone receptor, oncogenes, bassment membrane,
mesenchaymel tissue marker)
 8: tumor markers (cell proliferation markers, lymphoid markers, trouble shooting)
 9: tutorial (1)
 10: Imunofourecent methods
 11: Imunofourecent techniques (pathological applications)
 12: Enzyme histochemistry (techniques)
 13: Enzyme histochemistry (pathological application)
 14: overview of in-situ hybridization
 15: In-situ hybridization methods (probe preparation, pretreatment of specimens,
hybridization) detection methods



7. Immunohistochemistry

8.  IHC takes its name from the roots "immuno," in
reference to antibodies used in the procedure,
and "histo," meaning tissue (compare to
immunocytochemistry). The procedure was
conceptualized and first implemented by Dr.
Albert Coons in 1941
 Definition
 IHC is a method for demonstrating the presence
and location of (protein) cellular or tissue
constituent (as antigens), by mean of antigen

9. How
 Immunohistochemistry is the localization of
antigens in tissue sections by the use of labeled
antibody as specific reagents through antigen-
antibody interactions that are visualized by a
marker such as enzymes.

10.  It is accomplished with antibodies that recognise
the target protein .and this Ab bind only to the
protein of interest in the tissue section
 The aim is to achieve reproducible and
consistent demonstration of antigens with the
minimum background staining whilst
preserving the integrity of tissue architecture.

11.  For optimal staining to occur it is necessary to
use the primary specific antibody at the correct
dilution
 Blocking background staining (The major
causes of background staining in
immunohistochemistry are ionic interactions
and endogenous enzyme activity and
unbounded antibodies)…Endogenous enzyme
block – protein blocking -washing
 Unmasking of antigen ……antigen retrieval
Electrostatically charged slide

12. Incubation
 to prevent evaporation of antibodies, incubations
must be carried out in a moist atmosphere.
 Using the wax pen around the section to assist
with retaining the reagents on the section

13. steps
-Preparation of sample (retrivation)
-Immunohistochemical reaction
-Visualizing the reaction
-Counter stain.

15.  Applications of IHC
 especially advantages as a diagnostic tool for
infectious disease
 It is useful for assessing the progression and
treatment of disease such as cancers
 Differentiate between types of malignant cells
 Detection of metastatic tumours