LEC#13 HISTOPATHOLOGY LEC MLS–3APROF. Dr. Marissa A. Cauilan S.Y.

2022-2023STAINING
PRINCIPLES OF STAINING: Xylene

STAINING
∙ Is not miscible with aqueous solutions andlowgradedalcohol
●Process whereby tissue components are made visible in
microscopic sections by direct interaction with a dye or staining
∙ Should therefore be subsequently removedwith
solution absolute alcohol, followed by descendinggradesofalcohol to prevent damage
and detachment of sections.
●Enhance contrast and visualization of cell or its components
1.Deparaffinization
●Colored compound is used to produce contrast based on
varying affinities of cell components -Immersing the paraffin section in xylene 2x

○ colored produced by the tissues or structure are not

-After cutting, tissue will be embedded in a paraffinblocksoit's
the actual color of the tissue itself. The color produced
important that we now remove the paraffin becauseparaffinis
is based on the AFFINITY of that structure to the dye.
not visible with the stain since stain is usually inaquariusor
alcoholic solution.
○ Basic dye has strong affinity for acidic structure like
nucleus, hence, basic dyes stain the nucleus blue. In
-We have to remove the paraffin using xylene. It will be
contrast, the acidic dye has greater affinity for basic
immersed in xylene two times followed by rehydration.
structures for example the cytoplasm. acidic dye will
2.Rehydration
stain the cytoplasm since cytoplasm has a greater
affinity for acidic dye. -Absolute alcohol followed by descending grades of alcohol

○ Basic dye → acid structure ○ Acid dye → basic structure - paraffin will be removed with xylene thenremovethexylene using alcohol
and replacing it with water

3.Staining
HISTOLOGIC STAIN
-Immersing the sample in dye solution 4.Dehydration

●Purified form of a coloring agent or crude dye that is generally

applied in aqueous solution -Increasing grades of alcohol
●Individual variation in properties of tissue constituents
produce variation in color 5.Clearing -Two changes of xylene

○ that is why it is important to have a contrast between the different stains to

enable to differentiate
between the structure MORDANT grades of alcohol kaya tinatawag siyangna

Alcohol-based stains: no need to immerse in water, just thedescending METHOD OF STAINING:

●Chemical compound that reacts with the stain to form an insoluble, colored precipitate on the tissue
and make staining
reaction possible ●Process of giving color to the sections by usingaqueousor
DIRECT STAINING
○ this mordant act as a bridge because it forms a
alcoholic dye solutions
precipitate so it forms a mordant dye “lake” complex

●One dye is used

○ Mordant will also make counterstaining and
decolorization possible because usually the dye per
●Basic dyes-positive charge (giving a greater affinityto
se can stain the tissue but once decolorized or
negatively charge structures)
counterstain the stain may be easily removed
therefore with the use of mordant it will intensify the
●Ex: Methylene blue
stain because it will form an insoluble colored
precipitate. INDIRECT STAINING

STAINING OF PARAFFIN SECTIONS: Paraffin wax

∙ Action of the dye is intensified by adding another agentor a MORDANT
which serves as a bridge betweenthetissue and the dye.
 ∙ Is poorly permeable to most staining solutions and

∙ Mordant combines with a dye to forma colored“lake”,

shouldtherefore be removed from the section prior to
staining. complex”.
whichin turn combines with the tissue to forma“tissue-mordant- dye-

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o This allows subsequent counterstaining and

*If the primary dye is a basic dye, the differentiator wouldbean
dehydration to be carried out easily. METACHROMATIC STAINING
acid solution while if the primary dye is an aciddye, thedifferentiator would itself.
be a basic solution.
o A mordant may be applied to the tissue before the stain or may include as o Example:
part of staining technique orit may be added to the dye solution
∙ It entails the uses of specific dyes which differentiateparticular substances
by staining themwithacolorthat is different from that of the stain itself.
Potassium alum with hematoxylin in
∙ Tissues components combine with these dyestoform
Ehrlich’s hematoxylin and iron in
∙ It is particularly employed for stainingcartilage,connective tissue,
epithelial mucins, mast cellgranules, and amyloids.

∙ Azure or toluidine blue

∙ The actual staining process may involveimmersingthe sample (before or

after fixation and mounting)indye solution, followed by rinsing and
observation.
Weigert’s hematoxylin
METALLIC IMPREGNATION
a different color from the surrounding tissue.
∙ Process where specific tissue elementsare
PROGRESSIVE STAINING
demonstrated,not by stains, but by colorlesssolutions
of metallic salts which are reduced by thetissue,
∙ A process whereby tissue elements are stained in a
producing opaque, usually black depositsonthe
definitesequence, and the staining solution is applied for
surface of the tissue or bacteria.
specific period of time or until the desired intensity of
coloring of thedifferent tissue elements is attained. o The most valuable metals for the purposesaregold(gold chloride)
and silver (silver nitrate)
o Once the dye is taken up by tissue, it is not washedor decolorized.
VITAL STAINING

∙ NOTE: The distinction of tissue relies solely on the

∙ Selective staining of living cell constituents,

selectiveaffinity of the dye for different cellular elements.
∙ Demonstrating cytoplasmic structures by phagocytosis
REGRESSIVE STAINING ●Tissue is first overstained of the dye particle (cytoplasmic phagocytosis), or bystaining of pre-
existing cellular components (truevital staining) --- staining of
mitochondria by Janusgreen
●Excess stain is removed or decolorized until desired intensity
of color is attained.
∙ Examples: (i.) vital staining of reticulo-endothelial system (with Tyrpan
●Hematoxylin and Eosin (commonly used) blue, or propidiumiodideforeukaryotic cells)

●Overstained the tissue with hematoxylin then, counterstain it

∙ Purpose: To reveal cytological details (Note: nucleus
with eosin of a living cell is resistant to vital stain that’swhyisNOT demonstrated)
DIFFERENTIAL STAINING
∙ Note: “Demonstration of nuclear structures duringvital
●Selective removal of excess stain from the tissue during
staining suggests permeability of the membraneof the
regressive staining
∙ Injecting the dye into any part of the animal body(either intravenous,
dye,signifying the death of the cell.”
intraperitoneal or subcutaneous),
●Uses more than one chemical stain to better differentiate between various ●Requires washing or use of acids and oxidizing agents ●Basic dye-acid
microorganisms or structures/cellular
INTRAVITAL STAINING solution

producing specific coloration of certaincells,particularly those of the

components reticulo-endothelial system.

∙ Common dyes used:

●Alcohol acts as a differentiator for both basic and acidic dyes

●Mordant can act as a differentiating agent ●The differentiating solution is o lithium, carmine and India ink

important to produce different

contrast of colors on the tissue that is why it was called differential staining.

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SUPRAVITAL STAINING
●Cellulose nitrate (celloidin) is soluble in absolutealcohol and
will be removed if absolute alcohol is usedinthefinal
∙ Used in microscopy to examine living cells that have
been removed from an organism.
∙ Note: (partly) Due to their toxic interaction inside a living cell, when
dehydration prior to clearing of stained sections.
supravital stains enter a living cell, they might produce a characteristic
pattern of staining
∙ Supravital stains: those that enter and stain living cells (examples: New
Methylene Blue and Brilliant Cresyl Blue for reticulocyte staining --- toxic to STAINS AND STAINING SOLUTIONNATURAL DYES
the organism)

- natural dyes are those obtained fromplants andanimals,

different from the staining of an already fixed cell
("reticulocyte" look versus diffuse "polychromasia") natural dyes available are:
previously utilized for dyeing of wool and cotton. Amongthemost common

∙ Stains are used in very dilute solutions --- ranging from

1:5,000 to 1:50,000 ● natural dye derived by extraction fromthecoreor
1.Hematoxylin
o Note: Many stains may be used in both living
and fixed cells. the heartwood of a Mexican treeknownas"Hematoxylin
Campechianum”.
∙ Common dyes used are: 1. Neutral red - probably the best
● most valuable staining reagent ● Powerful cytology stain
vital dye. because it haspowerful

2. Janus green - especially recommended for

nuclear and chromatin staining capacity.
mitochondria. ● polychrome property
3. Trypan blue - one gram of dye is dissolved in 100 ml.
● Hematin – active coloring agent
of sterile distilled water to be used immediately; it is
dangerous to allow the suspension to stand for more
● most frequently used in combination withalum, iron,
than one hour, because it is likely to become toxic to the
cell. ● Without the mordants we cannot maximize the stain/dye.

4. Nile blue 5. Thionine 6. Toluidine blue ● We need these mordants so that when youdecolorizeacounter stain, the
structures will still retain thecolorofHematoxylin dye.
chromium, and copper salts, which act asmordants.Note:
HEMATOXYLIN AND EOSIN STAINING

● extracted from the female cochineal bug(Coccus

∙ Corner stone of tissue-based diagnosis
2.Cochineal Dyes
∙ To stain cell nuclei (and other parts) blue and an eosin
dyeto stain other structures pink or red Cacti), which is treated with alumto producethedye,carmine.
∙ Binds strongly to acids and consequently binds to
nuclear DNA and stains nuclei blue preparations (best for cytology).
● powerful chromatin and nuclear stain for freshmaterial and smear
∙ Most fixatives can be used except osmic acid
solutions which inhibit hematoxylin (neuropathological studies)
● Best’s Carmine stain (glycogen) ● Picocarmine
All specimens are initially stained using H&E (reason: H&E can confirm the basic tissue type and help to localize
the 3.Orcein
lesion. Thus, “lesion” means any area of damage, infection,
inflammation, tumor, necrosis or otherwise abnormal tissue) FROZEN ● vegetable dye extracted fromcertainlichenswhichare normally colorless,
but which, whentreatedwithammonia and exposed to air, produceblueor
SECTION STAINING violet
●stained as in paraffin sections although the duration of
staining is usually shorter. COLLODIONIZATION OF SECTIONS

●produces well-differentiated sections that are semi-permanent. colors.

● weak acid and soluble in alkali ● mainly used for staining elastic fibers.

●process of coating the slide with dilute (thin) celloidin solutions.

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SYNTHETIC DYES
∙ Romanowsky dyes
Ex:
Source: derived from hydro-carbon benzene (C6H6) taken
from coal tar
∙ Giemsa’s stain
-also known as “Coal Tar Dyes” or “Aniline Dyes” Chromophores
∙ Irishman’s stain
COMMON STAINING SOLUTIONS HEMATOXYLIN

-are substances with definite atomic groupings and are

capable of producing visible colors. Chromogens ●mordants used to demonstrate nuclear endcytoplasmicstructures are alum
and iron, forming lakes or coloredcomplexes
●most commonly used for routine histologic studies
-are simple benzene compounds that contain chromopores
Auxochrome ●basic dye
-an auxiliary radical or substance which imparts to the
●stain acidic (or basophilic) structures purplish blue
compound the property of electrolytic dissociation, thereby
altering the shade of the dye, enabling it to form salts with
COMMON STAINING SOLUTIONS
another compound, and ultimately retaining its color.
Note:
ALUMINUM HEMATOXYLIN SOLUTIONS
∙ Chromogens are different from dyes in that any color
that they impart to the tissue is not permanent and can,
therefore, be easily removed. ●progressive and regressive staining

●aluminum salts give a blue lake, and increasetheselectivityfor nuclei,

1. Acid dyes
Active coloring substance is found in acid component andinactive base
Ex: Picric acid- forms salt with an alkaliPicric acid
-is the only substance so far that can fix, differentiate and
stain tissue all by itself
usually passed on to an alkaline solution (e.g. 1%hydroxide)inorder to
-act as counterstain to basic cytoplasmic stains, to acid
fuchsin in Van Gieson's connective tissue staining, or
to crystal violet for the microscopic study of fungi. --used as fixative
-used as decalcifying agent
-used as tissue softener
Acidophilic-refers to basic cell structures (collagen, eosinophilic
especially if acid is added or is usedasadifferentiating agent
●Ehrlich’s hematoxylin and Harris hematoxylin solutions
●During staining, alum hematoxylin stained sectionsare
neutralize the acid and free the OHgroup, toforman
insoluble blue aluminum hematin-tissue-lake. Suchprocedure
granules of leukocytes, etc.) that have anaffinity for the acid dye ions. 2.
Basic Dyes
active coloring substance is found in a basic component that combines
with the acid radical
is known as blueing HEMATOXYLIN

●Blueing with ammonia, lithium carbonate or ●hematoxylin 2 gm ●absolute ethyl alcohol 100 m ●aluminum potassium
Scott’sTapWaterSubstitute has more rapid action than tap water
sulfate 15 gm approximately
○tap water is enough to produce blueing of the hematoxylinEHRLICH’S

(usuallytaken from sulfuric, acetic or hydrochloric

acid). ●distilled water 100 ml
Ex: Methylene blue
●glacial acetic acid 10 ml
Methylene blue
-for bacterial staining ●regressive staining, and differentiated with 1%hydrochloricacid in 70%
-both used as indicator and dye Basophilic alcohol (acid-alcohol)
-refers to acidic cell structures (chromatin, mucus, ●mucopolysaccharide substances such as cartilageand
●glycerin 100 ml
cartilagematrix etc.) have an affinity for basic dye ions
3. Neutral Dyes acid decalcification
cement lines of bones are also stained intensely blue●tissues subjected to

-formed by combining aqueous solutions of acid

andbasic dyes, ●not an ideal for frozen sections
-insoluble or barely soluble in water but soluble in alcohol - Ethyl alcohol or acetic
acid-fixed tissues, on the otherhand, readily take in both basic and acidic dyes.

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HARRIS HEMATOXYLIN
-Absolute ethyl alcohol ml

●hematoxylin 1 gm SOLUTION B:

absolute ethyl alcohol 10 m ammonium/potassium alum 20 gm -30% anhydrous ferric chloride 4 ml

distilled water 190 ml -Concentrated hydrochloric acid 1 ml
mercuric oxide (red) 0.5 gm glacial acetic acid 10 ml
-Distilled water 100 ml
●good regressive stain HEIDENHAIN’S HEMATOXYLIN
●routine nuclear staining, in exfoliative cytology, and for
staining of sex chromosomes grey - black.
●regressive staining of thin sections - all componentsareblackor dark
COLE’S HEMATOXYLIN
●nuclear and cytoplasmic inclusions such as chromatin,
●hematoxylin 1.5 gm chromosomes, nucleoli, centrosomes, and mitochondria.

1% Iodine in 95% alcohol 50 ml sat. aq. ammonium alum 700

●Voluntary muscle striations and myelin
ml distilled water 250 ml -MORDANT DIFFERENTIATOR: Ferric
●alum hematoxylin solution recommended for routine
ammonium sulfate 2.5 gm
purposes -Distilled water 100 ml
●artificially ripened with an alcoholic iodine solution
-HEMATOXYLIN STAIN: Hematoxylin 1.5 gm
MAYER’S HEMATOXYLIN
-95% ethyl alcohol 10 ml
●hematoxylin 1 gm sodium iodate 0.2 gm potassium alum 50
-Distilled water 90 ml
gm citric acid 1 gm chloral hydrate 50 gm distilled water 1000
ml PHOSPHOTUNGSTIC ACID HEMATOXYLIN(PTAH)

●chemically ripened with sodium iodate ●regressive stain and
progressive stain
●nuclear counterstain to demonstrate the presence of
cytoplasmic glycogen ●mucopolysaccharides
●Nuclei, fibrin, muscle striations, and myofibrils arecoloredblue while
collagen, bone and cartilage take anorange-redorbrownish red to deep
brick-red stain.
●celestine blue hemalum method of nuclear staining IRON HEMATOXYLIN

●Progressive SOLUTIONS

-Hematoxylin 1 gm -Phosphotungstic acid 20 gmDistilled Water 1000ml

EOSIN

●most valuable stains used for differentially stainingconnective

●differential or regressive staining, using acid-alcohol as a
differentiating agent. tissues and cytoplasm ●acid dye/acidophilic/ oxyphilic/ eosinophilic

●Weigert's Solution, using ferric ammonium chloride, and Heidenhain's solution, using ferric
ammonium sulfate (iron
alum) as mordants ●counterstain to hematoxylin ●background stain

●Tissue structures are stained blackish or grayish, according to

the extent of differentiation ●Eosin Y and Eosin B
REGAUD’S HEMATOXYLIN ROMANOWSKY STAIN
●demonstrate mitochondria by light microscopy ●based on a combination of eosinate (chemicallyreducedeosin) and
methylene
WEIGERT’S HEMATOXYLIN SOLUTION
●Wright's stain, Jenner's stain, Leishman stainandGiemsa
●standard iron hematoxylin stain used for demonstrating
muscle fibers and connective tissues ●blood or bone marrow samples
stain
●recommended when the preceding stains contain acid (e.g.
Van Gieson stain containing picric acid) which decolorizes
●blood-borne parasites like malaria.
nuclei stained with alum hematoxylin
- Used in hematology and microbiology
SOLUTION A:

-Hematoxylin 1 gm

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OTHER STAINS 4.Mallory’s Fuchsin Stain

3.Schiff’s Reagent

Acid Fuchsin-Picric Acid (Van Gieson’s Stain)

5.Aldehyde Fuchsin (Gomori’s Stain)

●mixture of picric acid and acid fuchsin for demonstration of ●stain collagen, smooth muscle, or mitochondria
BENZIDINE
connective tissues. ●For staining hemoglobin

Acid Fuchsin (Masson Stain) CARMALUM (MAYER'S) SOLUTION

●mordanted dye acting as a basic dye and stainingacidic

●nuclear and cytoplasmic stain in Mallory's trichrome method substances

●Van Gieson's picro-fuchsin, acid fuchsin imparts its red color ACRIDINE ORANGE
CELESTINE BLUE
to collagen fibers ●oxazine dye used as an alternative to ironhematoxylinnuclear stain

●permits discrimination between dead and living cells

(discrimination between dead and living cells is used in
combination with ethidium bromide)
●green fluorescence for DNA and a red fluorescence for RNA
●nucleic acid selective fluorescent cationic dye useful for cell
cycle determination
●forms a strong staining lake with iron alum, actingasa
mordant to bind hematoxylin
●resistant to strong acid dyes, and is recommendedfor routine
staining of fixed sections, giving a good nuclear definitionwhen
used in conjunction with alum hematoxylin.
ACRIDINE RED 3B
CONGO RED

●is best known as an indicator

●demonstrate deposits of calcium salts and possible sites of

●used to identify deposits of protein in tissue calledamyloid.
phosphatase activities CRESYL VIOLET

ALCIAN BLUE ●stain nervous tissues

●complex, water-soluble phthalocyanin dye, similar to
●stains the acidic components of the neuronal cytoplasm
chlorophyll, which stains acid mucopolysaccharides by forming
salt linkages with them ●blue color resistant to various counterstaining procedures
(specifically Nissl bodies) a violet colorCRYSTAL VIOLET

●is a nuclear or chromatin stain used for stainingamyloidin

●specific for connective tissue and epithelial mucin
 frozen sections and platelets in blood
●often combined with PAS ALIZARIN RED S methyl violet and dexterin.

● forms an orange-red lake with calcium at a pH of 4.2

●Gentian violet - staining solution formed by themixtureofcrystal violet, ETHIDIUM BROMIDE

ANILINE BLUE ●intercalates and stains DNA, providing a fluorescent red-orange stain
●cytoplasmic stain used for counterstaining of epithelial
sections. ●used in conjunction with acridine orange (AO) inviablecellcounting - live
cells to fluoresce green whilst apoptoticcellsretain the distinctive red-
AZOCARMINE orange fluorescence.

●Nuclei are deep red, cytoplasm is pale red BASIC FUCHSIN ●deep staining of acid- fast organisms, for mitochondria, for differentiation of
smooth muscles with the use of picric acid
●marker for apoptosis in cells populations ●locate bands of DNA in GIEMSA STAIN
gel electrophoresi

●mixture of methylene-blue and eosin

●Feulgen's and Schiff's reagent for the detection of aldehydes
●staining blood to differentiate leukocytes
●Van Gieson's solution for connective tissues, mucin, and for
elastic tissue staining 1.Carbol-Fuchsin 2.Coleman’s Feulgen Reagent ●It also binds to some pathogens, includingspirochetes(syphilis),
trypanosomes (sleeping,
●mostly used on methanol-fixed blood films

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sickness and Chagas disease) and plasmodium(malarial

homologues of the dye (azures) and deaminizedoxidation
parasite). products (thiazoles).

GOLD SUBLIMATE ● methylene blue - methylene blue, azures andthiazoles.

●stain used for metallic impregnation, made up of gold chloride

● stains nuclei blue while cartilage matrix, mucin, mast cell
and mercuric chloride. granules and connective tissues generally take areddish-violetcolor.

IODINE ●Plasma cells

●stains amyloid, cellulose, starch, carotenes and glycogen
●cytological examinations of fresh sputumfor malignant
●widely used for removal of mercuric fixative artefact pigments,
cells
and as a reagent to alter crystal and methyl violet so that they
may be retained by certain bacteria and fungi. ●aqueous or alcoholic ●diagnosis of diphtheria

solutions GRAM’S IODINE ●vital staining of the nervous tissue NILE RED●formed by boiling Nile blue

●used to identify and differentiate bacteria with sulfuric acid

●bacterial

● Gram-positive - staphylococci, streptococci and

● lipophilic stain; it will accumulate in lipidglobules
pneumococci gram-positive inside cells, staining them red OIL RED O

●Gram-negative - coliforms and Neisseria LUGOL’S IODINE ●multiple sclerosis - macrophages take upthelipid-richdebris and stain
strongly with this dye
● brown solution that turns black in the presence of
starches ● identify neutral lipids and fatty acids insmearsandtissues

●mordant in Gram's staining ●Fresh smears or cryostat sections of tissue ●identifying fat emboli in

JANUS GREEN lung tissue or clot sectionsof

● used for demonstrating mitochondria during intravital

staining peripheral blood

MALACHITE GREEN ORCEIN

●excellent stain for elastic fibers

●weakly basic dye used as a contrast stain for staining ascaris
eggs and erythrocytes, and as a bacterial spore stain
●dermatological studies - ability to demonstratethefinest
and most delicate fibers in the skin
●used both as a decolorizer and as a counterstain
●primarily a counterstain ●selective stain for unsaturated lipidsandfor
OSMIUM TETROXIDE
MASSON’S TRICHROME
lipoproteins such as myelin
●three-color staining protocol ●Fat, which reduces osmium tetroxide to osmiumdioxide, isstained black,
and may be demonstratedfromthe
●distinguish cells from surrounding connective tissue
tissue by using chrome-osmium solutions or bythefrozen
section method
●produce red keratin and muscle fibers, blue or green staining
of collagen and bone, light red or pink staining of cytoplasm, and black cell PERIODIC ACID SCHIFF (PAS)
nuclei.

METHYL GREEN capsules, and blood vessels as well asfungi andintracellular carbohydrates
●stains glycogen, mucin, mucoprotein, glycoprotein, basementmembranes, such as glycogen in hepatocytes
●stains chromatin green in the presence of an acid

●false positive reactions with mucin METHYLENE BLUE PHOSPHOTUNGSTIC ACID

●contains some azures or methylene violet ●common negative stain for viruses, nerves,polysaccharides, and other
●Cells that secrete mucus biological tissuematerials.
● stains acidic cell parts (like the nucleus) blue and is a good
counterstain with Eosin Y ●demonstration of striated muscle fibersandmitochondria, which stain blue.
●"Polychroming" involves the oxidation of methylene blue, resulting in loss of methyl
groups and leaving lower
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PICRIC ACID azures, which are reddish purple)

●Azurophilia (affinity for the oxidation products of methyleneblue called
●contrast stain to acid fuchsin, for the demonstration of
connective tissue (Van Gieson's stain) ●cytoplasmic stain in contrast ●microanatomical color contrast of specimens ●demonstration of the blood

to basic dyes ●counterstain to crystal violet and lymph vessels by injection

●tissue fixative and decalcifying agent. PRUSSIAN BLUE

●Acidophilia ( affinity for eosin)

Sudan Black
●neutrophilia (affinity for a complex of dyes inthemixture,which are pale
lilac)
(intravital staining)
●Usually for Hematology
RHODAMINE B
●Most sensitive of the oil soluble dyes
OIL SOLUBLE DYES (LYSOCHROMES) ●greater affinity for phospholipids than other lysochromes-coloring neutral
● is an insoluble colored salt of ferric ferrocyanide (an iron cyanide lipids (triglycerides) by simple dissolutionof thedye.
compound)
●used with osmic acid to fix and stain blood and glandular
tissues ●has no secondary amino group ●does not color phospholipids or the fine

SAFRANIN lipid droplets

●produces red nuclei ●recommended for staining triglycerides ( neutral lipids), givingthem a
deep and intense red stain
●used primarily as a counterstain ●give a yellow color to collagen
●used in 10% aqueous solution to prepare various dilutions to be used in
Silver Nitrate identification of spirochetes, reticulum and other
Sudan III
●ability of fats to absorb Sudan Black is relatedtodyeconcentration,
temperature and physical state of thefats. fiber stains.
●good as a fat stain for central nervous systemtissues,
Sudan IV

●In Histopath it is more on the reticulum, which it stains black.

●giving a less deep and lighter orange stain comparedtothe

Toluidine Blue CHIEF SOLVENTS USED FOR STAINS

darker staining Sudan IV.
●It is recommended for staining of Nissl granules or
chromophilic bodies. 1.WATER - most common

●stains nucleic blue, and can be used to differentiate different

2.ALCOHOL - most common
types of granules ( e.g. within mast cells). 3.ANILINE WATER
●preliminary stain to identify sections that will later be
examined by electron microscopy. Van Gieson Stain
4.PHENOL

●binds to collagen in the extracellular matrix, staining it pink.

●Often it is combined with a stain for elastic fibers (elastic van Gieson) which stain black,
allowing the two major elements of connective tissue to be differentiated.

Victoria Blue

●used for demonstration of neuroglia in frozen sections. Von Kossa Stain

●silver reduction method that demonstrates phosphates and carbonates.

Wright Stain

●Basophilia (affinity for methylene blue)

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