A TERM PAPER ON

STAINING PROCEDURE
Submitted to the department of Human Anatomy, SHHT,
Federal University of Technology Akure (FUTA)

GROUP LIST
IROEGBU JOY DUBEM

FAMOOFO OLUWASEUN

OGUNMOLA ESTHER

OGUNSHOLA ISRAEL

ADEPOJU ABIB

ABIADE AFEEZ

NWACHUKWU PAULINUS

ADEBOLA WILLIAMS

BAMISAYE MOBOALJI

OJO INIOLUWA

ADELE OLAMIPOSI

OLAGUNJU ISREAL
 TABLE OF CONTENTS
1. Introduction
2. Conventional histological staining
2.1. Errors encountered in conventional histological
staining
2.2. Table showing methods of conventional histological
staining
3. Staining in immunochemistry techniques
3.1. Intro
3.2. ABC-method
3.3. APAAP-method
3.4. Background staining and artifacts in immunochemical
staining
 1. INTRODUCTION.
While conventional histological staining methods have been established for decades, some
for more than a century, immunohistochemical techniques are not yet routinely used in
forensic diagnostics. They are used, however, when specific problems occur. In such cases,
depending on the problem, routine diagnostics may be supplemented with specific
microscopic techniques, including electron microscopy, laser scanner microscopy, and laser
microdissection techniques, in order to isolate single cells or cell groups. For important
routine diagnostics, established standard histological staining methods are discussed here.
Basic information on immunohistochemical techniques and on the best-practice use of
immunohistochemical and other methods are mentioned only briefly and therefore do not
substitute reference to the specialist literature.
Immunohistochemical staining techniques, in particular the ABC method, the
APAAP method, and the TUNEL technique, are used to label defined antigens with
monoclonal and polyclonal antibodies. Commercially produced antibodies mostly originate
from mice, less frequently from rabbits. In these cases, a number of methodological and
technical nuances must be considered in order to gain usable results. The degree of
autolysis or putrefaction, the selection of fixation medium, fixation duration, incubation
period, and concentration of the selected antibodies can be crucial. Different methods of
antigen unmasking are significant in a number of immunohistochemical stainings.
The following chapter gives a general overview of staining, highlighting the most
important aspects, including potential sources of error and the recognition of typical
mistakes and artifacts.
2. CONVENTIONAL HISTOLOGICAL STAINING
Conventional histological staining methods, including stain selection for specific
situations, have long been established. Descriptions of the most frequently used staining
methods are shown in (Table 2.1).
2.1
stains Presented structures

Haematoxylin–eosin Acidophilic cytoplasm is red, basophil

(H&E) staining nuclei are blue, erythrocytes are red
Alcian blue Detection of acid mucopolysaccharides
Azan staining (azo Connective tissue staining (red): azo
carmine and aniline blue) carmine
 stains cell nuclei, erythrocytes, fibrin,
fibrinoid, acidophilic cytoplasm, epithelial
hyalin; Aniline blue (blue): collagen fibers,
fibrous hyalin, basophil cytoplasm, mucus
Best’s carmine stain Classified as a glycogen stain, but is not
specific; also stains mucus, fibrin, gastric
glands, and mast cell granules
Elastika van Gieson (EvG) Combined staining of collagen fibers (red)
and elastic fibers according to Weigert
(black and brown); cytoplasm, musculature,
amyloid, fibrin, and fibrinoid (yellow)
Elastin staining according Stains elastic fiber violet black
to Weigert
Iron stain (Prussian blue reaction) Stains trivalent iron, in particular
hemosiderin;
detection of iron deposits
Gomori’s stain Argyrophilic reticular fibers (silver)
Grocott’s stain Ideal fungal stain: fungal conidia, fungal
fibers stain black
Mallory’s stain Trichrome stain; collagen and reticular
connective tissue is light-blue, nuclei are
red, smooth musculature is violet, striated
musculature orange-red, mucus is blue
PAS (periodic acid- Stains carbohydrates, in particular
Schiff’s reagent) glycogen,
purple-red (magenta) and epithelial mucin
Periodic acid – silver Stains basal membranes, Alzheimer’s
plaques, and fungi black
Toluidine blue Detects striation of muscle fibers and
metachromatic substances

3. Immunochemistry techniques
The range of immunohistochemically displayed cell and tissue proteins includes, e.g.,
collagens, basal membrane components, hormones, cytoskeleton proteins,
glycoproteins of cell membranes, viral and bacterial
antigens, cytokines, and complement factors.
Unlike conventional histological staining methods,
immunohistochemical techniques are based on antigen–
antibody bindings, which can be affected by inappropriate
fixative selection and duration.