This document discusses various staining techniques used to differentiate tissues and cellular structures under a microscope. It describes the objectives of staining as revealing internal and external structures and producing specific chemical and physical reactions. The 3 major groups of staining are histological, histochemical, and immunohistochemical staining. Histological staining uses dyes to demonstrate tissue and cell relationships. Histochemical staining localizes specific tissue substances through chemical reactions. Immunohistochemical staining uses antibodies to detect phenotypic markers. The document outlines different types of stains, staining methods like direct, indirect and regressive staining, as well as procedures for staining frozen sections, paraffin sections, and broken slides.

1. PRINCIPLES OF STAINING
McLean, Charlotte
Ong, Domingo

2. Objectives:
• To differentiate the different staining techniques
• To reveal the presence of various internal and
external structures
• To produce specific chemical and physical reaction

3. STAINING
• is the process of applying dyes on the sections to see and
study the architectural pattern of the tissue and physical
characteristics of the cell.

4. Alcian Blue and PAS (intestine). A stain that combines the properties
of both Alcian Blue and Periodic Acid Schiff staining.
• On a chemical basis, certain parts of cells and tissues that are acidic
in character (e.g nucleus) have greater affinity for basic dyes, while
basic constituents (e.g cytoplasm) take more of the acid stains
• In general, microscopic examination is facilitated if two contrasting
stains are used;
• e.g hematoxylin which stains the nuclear detail, and eosin which
brings out the cytoplasmic detail of the cell and the tissues
architecture

5. Gomori Trichrome Blue is used to stain and identify muscle fibers, collagen and nuclei
3 MAJOR GROUPS OF STAINING
 1. Histological Staining
- Process whereby the tissue constituents are demonstrated in sections by
direct interaction with a dye or staining solution, producing coloration of the
active tissue component.
 - micro anatomical or histologic staining is used to demonstrate the general
relationship of tissues and cells with differentiation of nucleus and cytoplasm

6.  2. Histochemical Staining (HISTOCHEMISTRY)
- Various constituents of tissues are studied thru chemical reactions
that will permit microscopic localization of a specific tissue substance.
 Example:
 -Perls prussian blue reaction for hemoglobin
- Periodic Acid Schiff staining for Carbohydrates
Perls prussian blue

7. Confocal fluorescence micrograph of HeLa cells stained with monoclonal antibody against mitochondria
enzyme and Cy3-conjugated anti-mouse antibody (red);
rabbit polyclonal antibody to histones in DNA and Cy5-conjugated-rabbit antibody (blue).
 3. Immunohistochemical Staining
 - A combination of immunologic and histochemical
techniques that allow phenotypic markers to be
detected and demonstrated under the microscope,
using a wide range of polyclonal or monoclonal
fluorescent labeled or enzyme-labeled antibodies

8. Human cheek cells stained
with methylene blue Prostate section stained with Hematoxylin & Eosin
(cytoplasm, pink; nuclei,blue)
METHODS OF STAINING
 DIRECT STAINING
 - A process of giving color to the sections by using aqueous or
alcoholic dye solution
 - e.g methylene blue, eosin

9.  INDIRECT STAINING
 - A process whereby the action of the dye is intensified by
adding another agent; either mordant or accentuator
 "MORDANT"
 - serves as a link or bridge between the tissue and the dye,
to make the stainig resction possible.
 - e.g Potassium alum with hematoxylin in Erlichs
hematoxylin, and iron in Weigers hematoxylin.
 "ACCENTUATOR"
 - Does not participate in the staining reaction, but merely
accelerates or hastens the speed of the staining reaction by
increasing the staining power and selectivity of the dye.
 - e.g Lofflers methylene blue and phenol in carbol thionine
and carbol fuchsin

10. This is a section of bone marrow stained with
Weigert's haematoxylin and counterstained
with eosin
Modified Loeffler's methylene blue
staining of Rhodococcus jostii

11. REGRESSIVE STAINING
 - Tissue is overstained to obliterate the Cellular details, and the
excess stain is removed or decolorized from unwanted parts of the
tissue, until desired intensity of color is obtained.
Lung tissue stained with the H&E technique. Nuclei are darkly stained in this image

12. PROGRESSIVE STAINING
- A process whereby tissue elements are stained in definite
sequence, and staining solution is applied for specific periods of
time or until the desired intensity of coloring of the different tissue
elements is attained.
- It result to "diffused color and obscured details" due to difficulty of
producing sufficiently intense progressive staining of cell structure
without staining other parts
 - less favoured than regressive staining

13. DIFFERENTIATION (DECOLORIZATION)
-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.
-usually done by washing the section in simple solution (e.g water
or alcohol) or by use of acid and oxidizing agents.

14. -Use of specific dyes which differentiate particular substances by staining t-
hem with a color that is different from that of the stain itself (metachromasia)
-for cartilage, conmective tissue, epithelial mucin, mast cell granules, and
amyloid.
-metachromatic dyes are basic dyes belonging to the "thizine and
triphenylmethane" groups, such as:
•methyl violet or crystal violet
•cresyl blue
•safranin
•Bismarck brown
•Basic fuchsin
•Methylene blue
•Thionine
•toluidine blue
•Azure A, B, C
METACHROMATIC STAINING

16. • All metachromatic dyes are cations or basic whose peculiar
staining property dpends upon their tendency to polymerize.
• All tissue components shown metachromasia are large anionic or
acidic molecules containing large amount of sulfate, phosphate, or
carboxylic acid radicals.
• WATER is necessary for most metachromatic staining techniques,
and metachromasia is usually lost if the section is dehydrated in
ALCOHOL after staining.
• the major group of metachromatic tissues consists of acidic
polysaccharides that occur in ground substance of cartilage
(chrondoitin sulfate) and in connective tissue mucin (acid
mucopolysaccharide) that bind basic dyes.

17. •COUNTERSTAINING
- Application of different color or stain to provide contrast and background
to the staining of the structural components to be demonstrated.
•CYTOPLASMIC STAINS
•NUCLEAR STAINS
red yellow green
Eosin Y Picric acid Light green SF
Eosin B Orange G Lissamine green
Phloxine B Rose bengal
RED BLUE
neutral red Methylene blue
Safranin O Toluidine blue
Carmine
Hematoxylin
Celestine blue

18. METALLIC IMPREGNATION
Is a process where specific tissue elements are demonstrated , not by
stains, but by colourless solutions of metallic salts which are thereby
reduced by the tissue, producing an opaque usually black deposit on the
surface of the tissue or bacteria.
a metallic impregnating agent is different from a stain in that it is not
absorbed by the tissue, but is held physically on the surface as a
precipitate or as a reduction product in certain tissue components.
It is utilized for silver staining of nervous system and also used to
demonstrate reticulin.
e.g. gold (gold chloride)and Silver nitrate, most commonly used agent
for impregantion , can also function as staining agent.
ammoniacal silver is reduced by argentaffin cells (in melanin and
intestinal glands), that forms black deposit seen under the microscope.

19. VITAL STAINING
- Selective staining of living cell constituents, demonstrating
cytoplasmic structure by phagocytosis of the dye particle
(cytoplasmic phagocytosis).
a. INTRAVITAL STAINING
- Staining of living cell is done by injecting the dye inti any part of
animal body (either intravenous, intraperitoneal or subcutaneous),
producig specific coloration of certain cells, particularly those of the
reticulo-endothelial system.
-lithium, carmine and india ink

20. SUPRAVITAL STAINING
- used to stain living cells immediately after removal from the living
body.
-common dyes are:
Neutral red – best vital stain
Janus green – recommended for mitochondria
Trypan blue – 1 g of dye is dissolved in 100ml of sterile dis.
Water to be used immediately; dangerous to stand for more than 1
hr, for it is toxic to the cell.
Nile blue
Thionine
Toluidine blue

21. STAINING OF PARAFFIN SECTIONS

22. MATERIALS USED FOR STAINING
Coplin jar –
slotted jar holding from
5-9 slides.
Slotted staining
dishes – holding
from 5-19 slides, over
which different
solutions are poured.
Slides are placed on
end singly or in
staggered fashion, in
the arm.
Metal or glass
staining racks or
carriers
– holding from 10-30
slides upright.

23. 4 Staining Methods Commonly
Employed for Frozen Sections
a. Hematoxylin-Eosin Method
b. Thionine Method
c. Polychrome Methylene Blue Method
d. Alcoholic Pinacyanol Method
(used also for suparavital staining of MITOCHONDRIA and
primarily for color sensitization in photography)

24. Routine H&E staining in Paraffin Embedded
Section (Regressive Staining)
lRoutine Hematoxylin and Eosin (H&E) staining- the most common
method utilized for microanatomical studies of tissues
lUsing regressive staining which consists of overstating the nuclei,
removal of superfluous and excessive color of the tissue constituent by
'acid differentiation'.
lMost fixatives can be used EXCEPT OSMIC ACID SOLUTIONS which
inhibit hematoxylin

25. RESULTS:
-nuclei – blue to blue black
-Cytoplasm, proteins in edema fluid
– pale pink
-Karyosome – dark blue
-RBCs, eosinophilic granules, keratin
– bright orange-red
-Basophil cytoplasm, plasma cells &
osteoblast -purplish pink
-Cartilage – pink or light blue to dark
blue (depends on the type of stain
used, darkest with Erlichs
hematoxylin)
Calcium and calcified bone –
purplish blue
Decalcified bone matrix, collagen
and osteoid – pink
Muscle fibers – deep pink

26. H&E staining of frozen section for
Rapid diagnosis
(Progressive staining)
•Reagents for this rapid H&E stain are generally arranged in sequence
using a series of coplin jars.
•This method takes only 5-10 min
•Produces well differentiated sections that are semi-permanet and can be
stored.
•The remaning portion of tissue must be kept for routine procassing and
are made for comparison with frozen sections.

27. RESULTS:
•Cells nuclei, cytoplasmic inclusions and muscle striations stain black.
•Other constituents are colored according to counterstain.
Heidenhains Iron Hematoxylin Method
Hamster Esophagus fixed in Bouin's
Fluid and stained with
Heidenhain's Hematoxylin only, at 40x

28. Celestine Blue-Haemalum Sequence
Staining
Celestine blue
-is an oxazine dye used as an
alternative to iron hematoxylin
nuclear stain, producing a strong
and precise nuclear stain that is
resistant to decolorization by
succeeding acid stains and
solutions.
- forms a strong staining lake with
'iron alum', acting as a mordant to
bind hematoxylin
RESULT:
Cell nuclei – blue
Other constituents colored
according to the counterstain used
a section of bone marrow, from the same block, stained with the
Celestin blue - haemalum method and counterstained with eosin.

29. Mallorys Phloxine Methylene Blue Stain
- Known as Eosin-Methylene Blue Method (EMB) method
- Produce sharp nuclear stain
- Reveals marked differentiation the various structures in
the tissues, which should be fixed in Zenkers fluid.

30. COLLOIDIONIZATION OF SECTIONS
Collodionization
-Is the process of more firmly attaching of ribbons by coating
the slide with dilute (thin) celloidin solutions.
-recommended for sections that will be subjected to strong
alkaline or acid solutions and for tissues that contain
glycogen for demonstration.
- celloidin will be removed in the final dehydration with
absolute alcohol prior to clearing and mounting.

31. STAINING OF CELLOIDIN SECTIONS
Cellulose Nitrate (celloidin)
-is soluble in absolute alcohol hence, treatment should be
avoided with abs. Alcohol during dehydration and clearing of
stained sections.
- sections treated with 95% alcohol may be transferred to a
mixture of equal parts of chlroform, absolute alcohol and
xylene then treated with xylene and mounted to xam.

32. RE-STAINING OF OLD SECTIONS
- Immersed in xylene for 24 hrs or gently heated until mounting medium
begins to bubble
- Coverslip may then removed by lifting it with a dissecting needle.
- Section is then placed in xylene for 30 min to remove the remaining
balsam and then brought down to water.
- It is placed in a 0.5 potassium permanganate solution for 5-10 min,
rinsed in tap water and subsequently immersed in 5% oxalic acid for 5
min or until the section is decolorized.
- Wash it again in running tap water for another 5 min
- The section may then be restained with the appropriate staining
technique.

33. BROKEN SLIDES
-for immediate examination:
mounting a broken slide to
another clean xylene -moist
slide with a drop of mounting
media (Clarite or Permount)
may be sufficient.
- replacement of slide is not
available, the section (if still
intact) may be transferred to
another slide.
- the coverslip can be removed by soaking
in xylene, and placing the broken slide in the
incubator at 37C until all the mountant has
been removed.
-the whole slide is then covered with a
mixture of 6 parts butyl acetate and 1 part
durofix and left in the incubator for 30 min
until the mixture hardens into a film.
-using a sharp scalpel blade, the hardened
film is cut around the section, and the slide
is placed in cold water until the film and
section float off.
- The film containing section is mounted in a
clean glass slide, placed in the 37C
incubator until dry, washed gently with butyl
acetate, then washed well with xylene, and
mounted in Clarite or Permount.