HISTOPATHOLOGY
PRINCIPLES OF STAINING
Most of the cells are colorless or transparent.
Therefor histological section have to be stained
in some way to make cells to be visible.
STAINING
 a process whereby tissue components
are made visible in microscopic sections
by direct interaction with a dye or
staining solution.
NOTE: Many dyes will be required to use
mordant because when the excess dye solution is
wash away the mordant still remains.
Histologic stain purified form of coloring
agent or crude dye that is
generally applied in an
aqueous solution.
EXAMPLE:
 Nucleus –have
greater affinity
for basic dyes
 Cytoplasm- take
more of acid
stains.
Mordant a chemical compound
that reacts with the stain
to form an insoluble,
colored precipitate on the
tissue and make the
staining reaction possible.
H&E staining is a great majority of
routine histology because
it is quick, cheap, and
informative.
 Hematoxylin-
stains the nuclear
details
• Eosin- brings out
the cytoplasmic
detail of the cell
and tissues
architecture.
STAINING OF PARAFFIN SECTIONS
Paraffin wax is poorly permeable to
most staining solutions
and should therefore be
removed from the section
prior to staining.
The alcohol is finally
with water before the
actual staining. It is sum
up with sections to water.
If alcoholic alcoholic
stain is used, No need to
replace with water;
The tissue sections will
be transferred to
decreasing grades of
alcohol and it is termed
as sections to alcohol.
For the sections to
alcohol is replaced with
water. But the sections to
alcohol, the alcohol
doesn’t need to be
replaced by the water.
Xylene- is not miscible
with aqueous solutions
and low graded alcohol
and should therefore be
subsequently removed
with absolute alcohol
 HISTOPATHOLOGY
PRINCIPLES OF STAINING
followed by descending
grades of alcohol to
prevent damage and
detachment of sections.
“Sections to Water” –if the alcohol is finally
replaced with water before the actual staining
section is performed. The exact reverse of
impregnation process.
“Sections to Alcohol”-after deparaffinization
with xylene, the sections is transferred to
decreasing grades of alcohol, it is use and the
staining procedure is subsequently done unless
the tissue has been fixed in mercuric chloride
solution in which the section is taken to water.
• Sections may float off the slide during
staining if the slides are dirty or greasy
or if the sections have not been left in
the paraffin oven long enough to dry and
be fixed in the slide. Sections must be
left in the oven for the minimum of 30
minutes before they are finally stained .
Methods of Staining
Direct Staining:
is the process of giving color to the
sections by using aqueous or alcoholic dye
solutions.
Simple(direct) staining-
only use one dye which is washed away after
30-60 seconds prior to drying and
examination. The molecule that make up basic
dyes have positive charge.
The cell wall and cytoplasm of bacterial cells
have negative charge, the positively charged
dyes are attracted to the negatively charged
cells, that’s why this stain has the ability to
stick and color the cells.
e.x. Methylene blue
-this blue stain will color all cells blue
making them to stand out against the bright
background of the light microscope.
Indirect Staining:
-is the process whereby the action of
the dye is intensified by adding another agent
or a MORDANT to make the staining
possible.
E.x. Mordants( potassium alum w/
hematoxylin in Ehrlich’s hematoxylin and
iron in Weigert’s hematoxylin.
NOTE: A mordant can be applied to the tissue
for the stain or it may be included as part of
the stainining technique or it may be added to
the dying solution itself.
ACCENTUATOR –is does not participate in
the staining reaction but merely accelerates
the reaction
E.x. Accentuator(potassium hydroxide in
Loeffler’s methylene blue and phenol in
carbol thionine and carbol fuchsin.
Progressive Staining:
-is the process whereby tissue elements
are stained in a definite sequence, and the
staining solution is applied for specific
periods of time or until the desired intensity of
coloring of the different tissue elements is
attained.
NOTE: The differentiation or distinction of
 HISTOPATHOLOGY
PRINCIPLES OF STAINING
tissue detail relies solely on the selective
affinity of the dye for different cellular
elements.
Regressive Staining:
-the tissue is first overstained to
obliterate the cellular details and the excess
stain is removed or decolorized from
unwanted parts of the tissue, until the desired
intensity of color is obtained.
e.x. Hematoxylin and Eosin staining is the
most common method utilized for
microanatomical studies of tissues, using the
regressive staining
Differentiation(Decolorization):
-the selective removal of excess
stain from the tissue during regressive
staining in order that a specific substance may
be stained distinctly from the surrounding
tissues.
- a staining procedure that
differentiates or distinguishes.
- A staining procedure that differentiates or
distinguishes between types of bacteria is
termed as a differential staining technique.
Differential Staining:
-uses more than one chemical stain to
better differentiation between various
microorganisms or structures/ cellular
components of a single organism.
-carried out by an acid solution while the
alkaline medium is used for differentiation
after applying an acidic dye.
-is also used to detect abnormalities in
the proportion of wbc cells in the blood.
A mordant can act as a differentiating agent.
Mordants such as iron alum can also oxidize
hematoxylin to a soluble, colorless
compound, so that the tissue component
becomes decolorized.
ALCOHOL –acts as differentiator and
probably dissolving out excess dye.
E.x. of Differential Staining Gram staining
uses two dyes:
Crystal violet and fuchsin or
safranin (the counterstain) to differentiate
between Gram-positive bacteria (large
peptidoglycan layer on outer surface of cell)
and gram-negative bacteria.
Metachromatic Staining:
-this technique entails the use of specific
dyes which differentiates particular
substances by staining them with a color that
is different from that of the stain
itself(metachromasia).
- At its simplest, the actual staining process
may involve immersing the sample (before or
after fixation and mounting) in dye solution,
followed byrinsing and observation.
E.x. Methyl violets, crystal violets-not
considered the most effective on purpose.
Azures or toluidine blue -are more effective
usually.
Amyloid- is exception ,it deposits amyloid
using crystal or methyl violets.
Metallic Impregnation:
-are demonstrated not by stains but by
colorless solutions of metallic salts which are
thereby reduced by the tissue producing an
opaque usually black deposit on the surface of
 HISTOPATHOLOGY
PRINCIPLES OF STAINING
the tissue or bacteria.
-agent is different from the stain in that
is not absorbed by the tissue, but held
physically on the surface as a precipitate or as
a reduction product in certain tissue
components.
Note: the most valuable metal are gold and
silver.
- Solutions should never be exposed to
sunlight if explosion is to be avoided, and all
unused reagents should be immediately
inactivated by sodium chloride or dilute
hydrochloric acid solution and discarded.
E.x. Ammoniacal silver –reduced by
argentaffin cells forming black deposits seen
under the microscope. This solution are
potentially explosive, care should be taken to
prepare all solutions in clean containers just
before use and silvered glassware should be
avoided.
Vital Staining:
- is a selective staining of living cell
constituents, demonstrating cytoplasmic
structures by phagocytosis of the dye
particle(cytoplasmic phagocytosis) or by
staining of pre-existing cellular components
(true vital staining) as in the staining of
mitochondria by Janus green.
-signifying the death of the cell.
- The usual purpose is to reveal cytological
details that might otherwise not be apparent;
however, staining can also reveal where
certain chemicals or specific chemical
reactions are taking place within cells or
tissues.
E.x. Trypan blue or propidium iodide for
eukaryotic cells (cells that has nucleus or
nuclear iodine)
Intravital Staining:
-is done by injecting the dye into any
part of the animal body (either intravenous,
intraperitoneal or subcutaneous)
NOTE: producing specific coloration of
certain cells, particularly those of the reticulo-
endothelial system.
E.x. Lithium, Carmine and India Ink
• Supravital Staining
-is a method of staining used in
microscopy to examine living cells that have
been removed from an organism.
-stains are used in a very dilute solutions
ranging from 1:5,000 to 1:50,000
E.x. New Methylene Blue and Brilliant
Cresyl blue for reticulocyte staining.
Common dyes :
*Neutral red-probably the best vital dye.
*Janus green-especially recommended for
mitochondria
*Trypan blue-one gram of dye is dissolved in
100 ml. of sterile distilled water to be used
immediately ;suspension stand more than one
hour becomes toxic to the cell.
*Nile blue
*Thionine
*Toluidine blue
• Hematoxylin and Eosin (H&E)
Staining
-the corner stone of tissue-based
diagnosis. In histology laboratory all
 HISTOPATHOLOGY
PRINCIPLES OF STAINING
specimens are initially stained with H&E and
additional stains are only ordered if additional
information is needed to provide a more
detailed analysis.
NOTE: Staining with H&E is very reliable
although it does show some variation
depending on the exact formulation of the
stain, and the stain density is considerably
affected by the thickness of the sections –
thicker sections take up more stain. It is also
generally done before any additional staining
techniques, because histology with H&E can
confirm the basic tissue type and help to
localize the lesion.
Procedure;
1.Clear paraffin embedded sections in first
xylene bath for 3 minutes.
2. Transfer to second xylene bath for 2
minutes.
3.Immerse in first bath of absolute ethyl
alcohol for 2 minutes
4. Transfer to a bath of 95% ethyl alcohol for
1 or 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 running tap water to remove excess
stain.
8. Differentiate in 1 % acid –alcohol(1 ml
concentrated HCL to 99ml of 80% ethyl
alcohol ) for 10-30 sec. monitoring the
changes in color microscopically until only
the nuclei are stained.
9. Rinse in tap water
10.Blue ammonia water (average of 5
minutes) or 1 % aqueous lithium carbonate
until the sections appear blue (about 30
seconds.)
11. Wash in running water for 5 minutes.
12. Counterstain with 5 % aqueous eosin for 5
minutes. If alcoholic eosin is use, 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 tp
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
-frozen sections mounted on the slides
may be stained as in paraffin sections
although the duration of staining is usually
shorter.
-may be stained by picking up sections on
albuminized slides and drying them quickly or
by simple direct staining on a wet slide with
an eye dropper.
E.x.
 Hematoxylin –Eosin Method
 Thionine method
Polychrome Methylene Blue Method
 Alcoholic Pinacyanol
method( used also for supravital
staining of mitochondria and
primarily for color sensitization in
photography )
Precautions in Staining
Take Note:
 Stains on skin should be avoided
because stains are health hazards.
 HISTOPATHOLOGY
PRINCIPLES OF STAINING

 Stains may be effectively removed with the appropriate staining technique.

from the skin by prompt topical
application of 0.5% acid alcohol,
followed by rinsing with tap
water.
 Failure of staining may be due to Histochemical
paraffin, fixative, or decalcifying Staining(Histochemistry)
solution that has not been
-a process whereby various constituents
thoroughly washed out and
of tissues are studied thru chemical reactions
removed.
 Stains may be saved and used that will permit microscopic localization of a
again for as long as they have not specific tissue substances.
lost their staining properties. E.x. Perl’s Prussian blue reaction for
Okay ra I re-use ug balik ang staining if, ang hemoglobin
iyang effect is still effective sa atong tissue.
Mas better gyud nga bag o ang stains. Periodic Acid Schiff staining for
carbohydrates
Collodionization of Sections
Immunohistochemical (IHC)
They are more firmly attached by coating
Staining
the slide with dilute (thin) celloidin
solutions, a process known as -a combination of immunologic and
collodionization, which is also histochemical techniques using a wide range
recommended for sections that will be of polyclonal or monoclonal ,fluorescent
subjected to strong alkaline or acid labeled or enzyme-labeled antibodies to detect
solutions and for tissues that contain and demonstrate tissue antigens and
glycogen for demonstration. phenotypic markers under the microscope.
-widely used in diagnosis of abnormal
cells such as those found in cancerous tumors,
Re-staining of Old Sections
in the localization of biomarkers, proteins in
Old, bleached or faded sections may be re- different parts of biological tissues,
stained: the slide is usually immersed in characteristics of cellular events such as
xylene for 24 hours, or gently heated until the proliferation or cell death(apoptosis).
mounting medium begins to bubble. The
ADDITIONAL: The current recommendation
coverslip may then be removed by lifting it
for immunohistochemical techniques is a
with a dissecting needle. The section is placed
maximum of 4% neutral buffered
in xylene for up to 24 hours to remove the formaldehyde solution and, for some
remaining balsam and then brought down to antibodies, fixation time can be up to a
water. It is placed in a 0.5 potassium maximum of 48 h.
permanganate solution for 5-10 minutes,
rinsed in tap water and subsequently
immersed in 5% oxalic acid for 5 minutes or
until the section is decolorized. After washing
it again in running tap water for another 5
minutes, the section may then be re-stained