PRINCIPLES OF STAINING possible.

By itself, the dye may stain only weakly,

if at all.
• STAINING
➢ Combines with a dye to form a colored
- Is the process whereby tissue components are
“lake”, which in turn combines with the
made visible in microscopic section by direct
tissue to form a “tissue mordant-dye-
interaction with a dye or staining solution.
complex” that is rendered insoluble in
- Produce a contrast between different tissues and
ordinary aqueous and alcoholic solvents.
cellular components based on their varying
- ACCENTUATOR is not essential to the chemical
affinities for most dyes and stains.
union of the tissue and the dye, it does not
- Is the purified form of a coloring agent or crude
participate in the staining reaction.
dye that is generally applied in an aqueous
solution. HISTOLOLOGICAL STAINING

STAINING OF PARAFFIN SECTIONS • PROGRESSIVE STAINING

- Is the process whereby tissue elements are
- Is poorly permeable to most staining solutions
stained in ta definite sequence, and the staining
and should therefore be removed from the
solution is applied for specific periods of time or
section prior to staining.
until the desired intensity of coloring of the
- Done by immersing the paraffin section in a
different tissue elements is attained.
solvent (e.g. xylene) two times, at 1-2 minutes
• REGRESSIVE STAINING
duration each, for sections up to 10 micron thick.
- The tissue is first overstained to obliterate the
- Xylene is not miscible with aqueous solutions and
cellular details, and the excess stain is removed
low graded alcohol and should therefore be
or decolorized from unwanted parts of the tissue,
subsequently removed with absolute alcohol,
until the desired intensity of color is obtained.
followed by descending grades of alcohol to
• DIFFERENTIATION (DECOLORIZATION)
prevent damage and detachment of sections.
- Is the selective removal of excess stain from the
- The alcohol is the finally replaced with water
tissue during regressive staining in order that a
before actual staining of section is performed.
specific substance may be stained distinctly from
- N is again dehydrated with increasing grades of
the surrounding tissues.
HISTOLOGICAL STAINING • DIFFERENTIAL STAINING
- Uses more than one chemical stain to better
- Is the process whereby the tissue constituents
differentiate between various microorganisms or
and general relationship between cell and tissue
structure/cellular components of a single
are demonstrated in sections by direct
organism.
interaction with a dye or staining solution,
• METACHROMATIC STAINING
producing coloration of the active tissue
- Technique entails the use of specific dyes which
component.
differentiate particular substances by staining
- Have developed many stains which are suited to
them with a color that is different from that of the
particular purposes, allowing cell structures to be
stain (metachromasia). Tissue components
differentiated.
combine with these dyes to form a different color
METHODS OF STAINING from the surrounding tissue.
• METALLIC IMPREGNATION
• DIRECT STAINING
- Is a process where specific tissue elements are
- Is the process of giving color to the sections by
demonstrated, not by stains, but by colorless
using aqueous or alcoholic dye solutions.
solutions of metallic salts which are thereby
• INDIRECT STAINING reduced by the tissue, producing an opaque,
- Is the process whereby the action of the dye is usually black deposit on the surface of the tissue
intensified by adding another agent or bacteria.
- MORDANT serve as a link or bridge between the
tissue and the dye, to make the staining reaction
• VITAL STAINING
- Is the selective staining of living cell constituents,
demonstrating cytoplasmic structures by
FIXATION: most fixatives can be used except osmic acid
phagocytosis), or by staining of pre-existing
solutions which inhibit hematoxylin.
cellular components (true vital staining), as in the
staining of mitochondria by Janus green.
• INTRAVITAL STAINING
- Is done by injecting the dye into any part of the
animal body (either intravenous, intraperitoneal
or subcutaneous), producing specific coloration
of certain cells, particularly those of the reticulo-
endothelial system.
• SUPRAVITAL STAINING
- Is a method of staining used in microscopy to
examine living cells that have been removed from
an organism.
COMMON DYES USED ARE:
1. Neutral red- probably the best vital dye.
2. Janus green- especially recommended for
mitochondria.
3. Typan blue- one gram of dye is dissolved in 100
ml. of sterile distilled water to be used
immediately; it is dangerous to allow the
suspension to stand for more than one hour,
because it is likely to become toxic to the cell.
4. Nile blue
5. Thionine
6. Toluidine blue
HEMATOXYLIN AND EOSIN (H&E) STAINING
- Is the corner stone of tissue-based diagnosis. The
process stains thin tissue sections so that
pathologists, can visualize tissue morphology.
- Process uses a hematoxylin dye to stain cell nuclei
(and other parts) blue and an eosin dye to stain
other structures pink or red.
- Staining plays a significant role in tissue-based
diagnosis by coloring otherwise transparent
tissue sections, and allowing cell structures
including the cytoplasm, nucleus, and other
organelles and extra-cellular components to be
clearly visible under the microscope.
ROUTINE H&E STAINING IN PARAFFIN EMBEDDED
SECTION (REGRESSIVE STAINING)
1. Clear paraffin embedded sections in first xylene
bath for 3 mins.
2. Transfer to second xylene bath for 2 to 3 minutes.
3. Immerse in first bath of absolute ethyl alcohol for
2 minutes.
4. Transfer to a bath of 95% ethyl alcohol for 1 to 2
minutes.
5. Rinse in running water for 1 minute.
6. Stain with Harris alum hematoxylin for 5 minutes
(Ehrlich’s hematoxylin requires 15-30 minutes)
7. Wash in running tap water to remove excess
stain.
8. Differentiate in 1% acid-alcohol (1ml
concentrated HCl to 99 ml. of 80% ethyl alcohol)
for 10-30sec. monitoring the changes in color
microscopically until only the nuclei are stained.
9. Rinse in tap water.
10. Blue in ammonia water (average of 5 minutes) or
1% aqueous eosin for 5 minutes. If alcoholic eosin
is used, the time can be reduced to 30 seconds or
1 minute.
11. Wash in running water for 5 minutes.
12. Counterstain with 5% aqueous eosin for 5
minutes. If alcoholic eosin is used, the time can
be reduced to 30 seconds or 1 minute.
13. If aqueous eosin is used, wash and differentiate
in tap water under microscope control until the
nuclei appear sharp blue to blue black and the
rest of the tissue appear in shades of pink. If
alcoholic solution is used, differentiate with 70%
alcohol.
14. Dehydrate, clear and mount.
 FROZEN SECTION STAINING HISTOCHEMICAL STAINING

1. Hematoxylin-Eosin method • Is the process whereby various constituents of

2. Thionine method tissues are studied thru chemical reactions that
3. Polychrome Methylene Blue Method will permit microscopic localization of a specific
4. Alcoholic Pinacyanol method (used also for supravital tissue substance.
staining of mitochondria and primarily for color
IMMUNOHISTOCHEMICAL (IHC) STAINING
sensitization in photography)

PROCEDURE: • Is a combination of immunologic and

histochemical techniques using a wide range of
1. Orient section in the block and freeze with liquid polyclonal or monoclonal, fluorescent labeled or
nitrogen. enzyme-labeled antibodies to detect and
2. Cut cryostat section at 5-10 micron. demonstrate tissue antigens (e.g. proteins) and
3. Mount sections on to albuminized slides and dip phenotypic markers under the microscope.
in 10% formalin to fix. • Widely used in the diagnosis of abnormal cells
4. Rinse rapidly in water. such as those found in cancerous tumors, in the
5. Stain with Harris hematoxylin for 30-45 seconds. localization of biomarkers and differentially
6. Rinse in tap water. expressed proteins in markers that are
7. Blue in ammonia water for 5 seconds. characteristic of particular cellular events such as
8. Rinse in tap water. proliferation or cell death (apoptosis).
9. Counterstain with 5% aqueous eosin or 1%
alcohol eosin for one minute.
10. Rinse in tap water.
11. Dehydrate in increasing concentrations of
alcohol.
12. Clear with xylene.
13. Mount with cover slide.

COLLODIONIZATION OF SECTIONS

• They are more firmly attached by coating the

slide with dilute (thin) celloidin solutions, is also
recommended for sections that will be subjected
to strong alkaline or acid solutions and for tissues
that contain glycogen for demonstration.

PROCEDURE:

1. Deparaffinize in xylene.
2. Dehydrate thru absolute alcohol.
3. Dip individual slides in Coplin jar containing dilute
ether alcohol solution.
4. Dip in dilute ether solution of celloidin (thin
celloidin).
5. Hold slide on one end for ½ to 1 minute to drain
or until the section begins to whiten around the
edges.
6. Wipe off the back of the slide and place in 80%
alcohol for 3-5 minutes to harden the celloidin.
7. Stain as desired.