This document provides information on various histological techniques used to process tissue samples, including fixation, processing, embedding, sectioning, and staining. It discusses the aims and methods of fixation, processing (dehydration, clearing, infiltration), embedding, sectioning, and different types of staining (histochemical, immunohistochemical, special stains). The staining techniques described include H&E, various histochemical stains for carbohydrates, lipids, nucleic acids, pigments, connective tissue, amyloid, and microbial organisms. Immunohistochemistry techniques are also briefly outlined.

2. Fixation of tissue
Processing of tissue
Histochemical stains
Immunohistochemistry
Quiz session

3.  Collections of :
› Special stains
› Immunohistochemistry
› Immunofluorescence
› Molecular studies
› Cytogenetic studies
› Electron microscopy
› Flow cytometry
› Polymerase chain reaction

4.  Simple.
 Low cost procedure.
 Fixation.
 Chemical processing.
 H&E stained slides.
 Rapid, comprehensive and informative scope.

5.  Aim of fixation:
› Nearest to its living state
› Change in size and shape
› Autolysis
› Firm to hard
› Bacterial growth
› Quality of cell
› Staining
 Media: 10% Neutral buffered formalin

6.  Aim of tissue processing:
› Sufficient rigidity
› Cut into thin sections
Dehydration
• Water molecule
is removed from
tissue
Clearing
• Dehydrating
agent is
replaced by
clearing agent
Impregnation
• Tissue is
infiltrated with a
supporting
medium

7.  Dehydration:
› Removes free or unbound water molecule.
› It is mandatory because the supporting media (paraffin) not miscible
with water.
› Media: Methanol or ethyl alcohol.
› Graded alcohol: 50, 70, 90 or 100%

8.  Clearing:
› Removal of dehydrating agent (e.g: alcohol) to facilitate infiltration
of paraffin wax.
› Make tissue clear and improves microscopic examination.
› Media: Xylene.

9.  Impregnation/infiltration:
› Clearing agent is removed by molten wax through process of
diffusion.
› Media: Paraffin wax

11. Tissue
processor
boxes with
their reagents

12.  Occur after processing.
 Processed tissue is put into
mould and paraffin wax is
poured.
 Aim:
› Give support to the tissue
› Prevent distortion of tissue
during cutting
› Preserve tissue for archival
uses
1
3
2

13.  Block is now prepared
 Tissue embedded blocks
are cut into thin sections.
Block
Thin section
ribbons
Block
1
2
3

14.  Tissue section is colourless, because the fixed protein has the
same refractive index as that of glass, thus stains are
required.
 Histochemical stain causes main chemical reaction that
demonstrate the tissue elements of interest.
 Stains have specific affinity with different tissue proteins and
color them differently.

15. • Hematoxylin and Eosin stain
Routine stain
• Carbohydrates
• Lipids
• Nucleic acid and proteins
• Pigments
• Connective tissue stain
• Amyloid staining
• Microbial organisms
Special
stains

16.  Introduction:
› Commonly used.
› Hematoxylin:
 Nuclear stain
 Bluish black
› Eosin:
 Cytoplasmic stain
 Pink
Hematoxylin
Eosin

18.  Mounting and coverslip
› Protective cover over the tissue
› Make a permanent bond between the
cover slip and slides.
› Fading of staining or any further
physical damage.

19.  Introduction:
› Demonstration of various cellular products like carbohydrates,
proteins, lipids and pigments for the diagnosis of certain diseases.
› Demonstration of extracellular material for example amyloid for
amyloidosis
› Identification of microbial organisms
› Estimation of DNA and RNA content of cell

20.  Classification:
› Carbohydrates
› Lipids
› Nucleic acid and proteins
› Pigments
› Connective tissue stain
› Amyloid staining
› Microbial organisms

21.  Carbohydrates:
› Periodic acid schiff (PAS) stain
› Alcian Blue stain
› Combined PAS and Alcian blue stain
› Mucicarmine stain

22.  Periodic acid schiff (PAS):
 Hydroxyl group (OH) of the
carbohydrate molecule is oxidized to
aldehyde (CHO) group by periodic acid.
 These aldehyde groups react with
Schiff’s reagent to form a magenta
coloured compound.

23.  Periodic acid schiff (PAS) application:
 Glycogen storage disorder
 Basement membrane of glomeruli and glands
 Capsule of various fungi such as cryptococci,
histoplasma, blastomycosis.
 Neutral mucin: Endocervical glands, intestinal
glands and bronchial glands
 Lysosomal storage disorder.
 Lipofuscin and pigments of Dubin-Johnson
syndrome.
 Russell bodies of plasma cells.
Lysosomal storage
disorder
Basement membrane
Fungal organisms

24.  Alcian Blue:
 Copper containing phthalocyanine ring in alcian blue is positively
charged which attracts anionic site of acidic mucin giving blue colour.

25.  Alcian blue:
› Barrett’s esophagus: Acidic mucin in
intestinal metaplastic cells.
› Mucinous adenocarcinoma in ovary:
Mucin.
› Pleural mesothelial cells: Hyaluronic
acid.
› Myxomas: Mucin secreting tumors.
› Discoid lupus erythematosus lesion
› Mucinous material in myxoedema
Intestinal metaplasia
Mesothelial cells
Mucinous secreting tumors

26.  Combined PAS and Alcian blue
principle:
 Demonstrate both acidic and neutral mucin
in single stain.
 Glycogen: Magenta colour
 Acidic mucin: Blue colour
Intestinal metaplasia
Fungal organism:
Cryptococcus

27.  Mucicarmine stain principle:
› Carmine complex of mucicarmine is positively charged and binds
with anionic mucin giving dark red colour.

28.  Mucicarmine stain application:
› Acidic mucin in intestinal
adenocarcinoma.
› Capsule of fungi such as
cryptococcus. Fungal organism:
Cryptococcus
Intestinal adenocarcinoma

29.  Lipids:
› Oil red O
› Sudan Black B
› Ferric haematoxylin for
phospholipid

30.  Oil Red O principle:
› Highlights lipid material gives deep red colour
› Precaution: Fresh tissue is always required.
› Reason:
 Stain is highly soluble in lipid substance
 If fatty tissue is stained with Oil red O and processed in alcohol.
 As fat is dissolved in alcohol solution along with oil red o stain, the
processed tissue will not retained.
 Therefore, fresh adipose tissue and aqueous mounting media is
required in replacement of alcohol solution.
 Liposarcoma
Liposarcoma

31.  Sudan Black B principle:
› Lipophilic stain
› Insoluble in water
› Dissolved in tissue fat and stains
dark black
› Stain is slightly basic – combines
with acidic component of lipid
› Fresh tissue is always required.
› AML in hematology and fat droplets
Fat droplets
AML

32.  Ferric Haematoxylin for Phospholipid
principle:
› Stains phospholipid and gives dark blue
colour
› Fresh tissue is always required
Nuclei stained dark blue

33.  In general, all lipid stains applied for:
› Lipids in renal cell carcinoma
› Lipoblasts in liposarcoma
Fat cells stained red

34.  Nucleic acid and proteins:
› Feulgen stain
› Methyl-green pyronine

35.  Feulgen stain principle:
› Specific for DNA
› Step 1: DNA + Hydrochloric acid
(acidic environment) – Aldehyde
group of deoxyribose are exposed.
› Step 2: Aldehyde group + Schiff’s
reagent – reddish purple colour.

36.  Methyl Green-Pyronine
Stain principle:
› Demonstrate both DNA and
RNA
› DNA stained bluish green.
› RNA stained red.
DNA stained bluish green
RNA stained red

37.  In general nucleic acid
and protein stains are
applied for:
› DNA ploidy examination

38.  Pigment stains:
› Hemosiderin pigment: Prussian blue reaction (Perl’s reaction) for
ferric iron.
› Bile pigment: Fouchet’s stain
› Argyrophil pigment: Grimelius stain
› Melanin pigment: Masson-Fontana stain and schmorl’s stain
› Calcium: Von Kossa stain
› Copper: Rhodanine stain

39.  Hemosiderin pigment: Prussian blue
reaction (Perl’s reaction) for ferric iron
principle:
› Hydrochloric acid unmask the ferric
iron.
› Ferric iron react with potassium
ferrocyanide to form insoluble blue
colored ferric ferrocyanide.
› Hemosiderin: Blue
› Nuclei: Red
Hemosiderin pigment in liver tissue
Perl iron
stain

40.  Bile pigment: Fouchet’s stain
principle:
› Bile pigments include
conjugated and unconjugated
bilirubin and biliverdin.
› Bile pigment: Green
› Collagen: Red
› Muscle: Yellow
Bile pigment

41.  Argyrophil pigment: Grimelius stain
principle:
 Argyrophil cells quickly absorb silver
salt and needs a reducing agent to
visible silver precipitation by
reduction
 Argyrophilic cells: Black
 Background: Golden yellow

42.  Melanin pigment: Masson-Fontana stain
and schmorl’s stain:
› Masson-Fontana stain:
 Demonstrate melanin and argentaffin
granules which stained black
 Nuclei: Red
› Schmorl’s stain:
 Demonstrate melanin and argentaffin
granules which stained dark blue
 Nuclei: Red
Malignant melanoma
Schmorl’s stain
Skin tissue: Melanocytes in the basal layer
Black pigment: Melanin

43.  Calcium: Von Kossa stain:
› Inorganic calcium is the
important constituents of bone
and teeth
› Calcium is deposited in
abnormally necrotic and
infarcted tissue
› Calcium deposits: Black
› Nuclei: Red
Dystrophic calcification in blood vessel wall

44.  Copper: Rhodanine stain:
› Stain yellow to brown pigment (copper) in Wilson’s disease
A: Copper pigment in hepatocytes. B: Rhodanine stain

45.  Connective tissue stain:
› Masson trichrome stain
› Verhoeff’s Van Gieson stain
› Reticulin stain
› Weigert’s Resorcin-Fuschsin stain
› Orcein stain
› Phosphotungstic acid Haematoxylin (PTAH)

46.  Masson trichrome stain:
 Stain cell components as follows
 Muscle: Red
 Collagen: Blue
 Nucleus: Black or blue
 Fibrin: Red
 Staining principle depends upon molecular
weight of cell.
 Larger molecular weight of cell: Less
penetration.
 Smaller molecular weight of cell: Easy
penetration.
 Thus, give different colours to tissue
accordingly

47.  Masson trichrome stain:
 Liver biopsy:
 To assess degree of fibrosis: Chronic liver
disease and cirrhosis
 Kidney biopsy:
 To see changes of basement of glomeruli
› Tumors:
 To distinguish collagen and smooth
muscle in tumors
Liver tissue: Cirrhosis (Blue
septas)
Basement membrane of glomeruli

48.  Verhoeff’s Van Gieson stain:
 Demonstrate
 Collagen: Red
 Nuclei: Black
 Cytoplasm, muscle and fibrin: Yellow.
 Extent of fibrosis: Quantification of
fibrosis for e.g: Atrophic fibers
 Tumors: Differentiate collagenous
material from smooth muscle to
differentiate fibrosarcoma from
leiomyosarcoma

49.  Reticulin stain:
 Reticulin fibers contain a carbohydrate
compound.
 Potassium permagenate oxidize reticulin
fibers.
 Aldehyde group is generated from
carbohydrate compound
 Silver salt in basic pH produces metallic
silver and reacts with aldehyde group
 Sodium thiosulphate removes excess water
 Gold chloride precipetate and gives black
colour to reticulin fibers while nuclei appear
red.

50.  Reticulin stain:
› Liver: Early cirrhosis
› Kidney: Kimmelstiel wilson lesion in diabetic
glomerulosclerosis
› Bone marrow: Marrow fibrosis and in myelofibrosis
› Lymph nodes: Non hodgkin lymphomas
Bone marrow
fibrosis
Liver architecture
KW nodules in diabetic
glomerulosclerosis

51.  Weigert’s Resorcin-Fuschsin stain:
› Stain elastic fibers and gives purple colour

52.  Orcein stain:
› Demonstrate fibrin and cross striations of muscle
› Elastic fibers: Brown

53.  Phosphotungstic acid Haematoxylin (PTAH):
 Stain fibrin and cross striations of muscle and glial fibers
 Striated muscle fibers, fibrin, nuclei and astrocytes: Blue
 Cytoplasm: Brown red
 Collagen and bone: Brown pink

54.  Amyloid staining:
› Congo red stain:
 Congo red intercalates between
parallel fibrils of the amyloid protein
and forms a non polar hydrogen
bond.
 Amyloid: Deep pink
 Apple green birefringence in
polarized light.

55.  Microbial organisms:
› Bacteria:
 Gram’s stain
 Ziehl Neelsen stain
 Fite Acid Fast stain
› Fungal:
 Grocott’s methenamine silver stain
› Spirochaetes:
 Warthin starry stain and giemsa stain
› Viral inclusions:
 Phloxine-Tartrazine stain

56.  Gram stain:
› Gram positive bacteria: Purple
› Gram negative bacteria: Pink
› Step 1: Crystal voilet (Cell wall is
stained)
› Step 2: Gram’s iodine binds with
crystal voilet to the peptidoglycan
cell wall: Crystal voilet iodide
complex.
› Step 3: Alcohol is added which
cleaves this complex.
› Step 4: Safranin stain is added.

57.  Ziehl Neelsen stain
› Demonstrates acid fast tubercular bacilli
› Organisms are stained with basic fuchsin
dye by heating
› Resist decolorization by acid alcohol
› Methylene blue (counterstain) stain
decolorized non acid fast bacilli.
› Acid fast bacilli: Red colour
› Nuclei: Blue colour
Acid fast bacilli: Red

58.  Fite Acid Fast stain
› Demonstrate mycobacterium leprosy
› Mycobacterium leprae bacilli are
weak acid fast organisms
› Weak concentration of acid fast
bacilli is used.
› Acid fast bacilli and mycobacterium
leprae: Red
› Background: Blue
M. Leprae

59.  Grocott’s methenamine silver stain:
› Cell wall of fungi: Black
› Outer wall of pneumocystis carinii: Black
› Background: Green
Fungal organisms: Cryptococcus and actinomycosis

60.  Warthin starry stain:
 Demonstrate spirochaetes: Black
colour
 Background: Brown to yellow
 Giemsa stain:
› Demonstrate helicobacter pylori:
Dark blue
› Background: Light blue

61.  Phloxine-Tartrazine stain:
› Viral inclusion: Bright red
› Background: Yellow

63.  Technique to visualize recognition of
antigen present in the tissue with the
help of corresponding antibody
 Antigen: Any substance capable of
producing an immunologic response.
Antigenic binding site is also known
epitope.
 Antibody: Any substance that has
specific affinity against the antigen. Also
known as immunoglobulin.

64.  Control:
› In any IHC staining, it is
extremely essential to have
proper control because it
validates the laboratory test.
Control slide indicate the
specificity of the test because it
is essential to know that the
antibody is reacted specifically
to the specific epitope of
particular antigen and not with
the other antigen.

65.  Detection system:
› It is not possible to detect antigen-antibody reaction by light
microscope. Therefore, suitable detection or visualization system
is necessary for demonstration of such reaction. Two important
types of detection system are as follows:
 Direct method
 Indirect method

66.  Direct Method:
› Primary antibody is directly tagged with an enzyme or
flourescence
› Antibody should be specific for particular antigen otherwise non
specific staining may occur.
› Rapid and simple method but has low sensitivity

67.  Indirect Method: (Routinely used
method in IHC)
› Primary antibody is unlabelled
› Secondary antibody is conjugated and
directed against the primary antibody
› Antigen-primary, antibody-secondary
complex is visualized by a chromogen
› Large amount of secondary antibody
can easily produced and has high
sensitivity

68.  Steps of IHC:
1) Antigen retrieval
2) Blocking of endogenous
enzyme
3) Blocking the background
staining
4) Incubation with primary
antibody
5) Incubation with labelled
secondary antibody
6) Visualization
7) Counter staining
8) Dehydration and mounting

70.  Before step 1: Deparaffinize (xylene) and rehydrate (graded alcohol)
 STEP 1: Antigen retrieval
› Formalin Fixed Paraffin Embedded (FFPE) tissues are previously processed
in formalin which causes enzymatic digestion of the tissue which causes
masking of antigen binding site by non specific antibody
› To overcome this issue “Antigen retrieval” is mandatory in FFPE tissue but not
in fresh tissue.
› Antigen Retrieval: Recovery of antigenicity of the tissue which is masked at
the time of formalin fixation.
› 3 methods:
 Microwave retrieval
 Pressure cooker heating
 Water bath heating

71.  STEP 2: Blocking of endogenous enzyme
 Reagent for blocking: 3% Hydrogen peroxidase in methanol for 10
minutes.

72.  STEP 3: Blocking the background staining
 Blood vessels and collagen in tissue
sections, may contain macrophages,
lymphocytes, monocytes or polymorphs.
 These cells contain Fc receptors which bind
with the antibody and may produce
background staining.
 Preincubation with 2% normal saline or
introduction of Fab fragments reduce
unwanted non specific binding.

73.  STEP 4: Incubation with primary antibody
 Optimal dilution of primary antibody is needed to avoid any false
negative staining.

74.  STEP 5: Incubation with labelled secondary antibody
 Secondary antibody is usually labelled and directed against the primary
antibody

75.  STEP 6: Visualization
 Appropriate chromogen is applied to visualize the reaction

76.  STEP 7: Counter staining
 Light counter stain (hematoxylin, 10s) is applied to visualize background
nuclei and tissue architecture

77.  STEP 8: Dehydration and mounting
 Dehydration: Alcohol
 Clearing: Xylene
 Mount: DPX

78. Nuclear Membraneous
Cytoplasmic

80.  A peripheral blood smear of a middle aged
man with cytopenias show these circulating
white blood cells that express CD103 by flow
cytometry. Which special stain will you
perform?
1. Reticulin stain
2. Periodic acid schiff (PAS) stain
3. Tartrate resistant acid phosphatase
(TRAP) stain
4. Melanin stain

81.  3) Tartrate resistant
acid phosphatase
(TRAP)
› Hairy cell leukemia –
Hairy cells show red
granular cytoplasmic
staining

82.  A 4 years old boy referred because of history of
blood in stools for 8 months. Before referral, a stool
culture was performed showed campylobacter jejuni,
adequately treated with antibiotics. Control culture
was negative. Diarrhea resolved but abdominal pain
and bloody stools persisted. Colonoscopic biopsy
was performed. Identify the pathology and order
special stain.
1. Intestinal spirochetosis - Warthin starry stain
2. Giardiasis – Giemsa stain
3. Celiac disease – CD3 IHC stain
4. Amoebic colitis – Periodic acid schiff stain

83.  1) Intestinal spirochetosis – Warthin starry stain

84.  A 15 year old boy, diagnosed case of Thalasemia
intermedia has been receiving multiple transfusions of red
blood cells since early childhood. On physical examination,
he has no significant findings except for mild hepatomegaly.
Which of the following special stain would be helpful on his
liver biopsy?
1. Alcian blue
2. Fontanna Masson stain
3. Perl stain
4. PAS stain

85.  3. Perl stain highlights hemosiderin pigment (blue) in liver
parenchyma.

86.  A 26 years old male resident of
Balochistan presented with a
nodular skin lesion with central
ulceration on his nose. Identify
the stain highlighting the
organism?
1. Gorocott methenamine silver stain
2. Giemsa stain
3. Periodic acid schiff stain
4. Warthin starry stain
H&E slide
Identify stain?

87.  2. Giemsa stain highlights Leishmania Donovan bodies

88. During the preparation of a routine H&E slide, what allows the
tissue to hold its form?
1. Fixation
2. Embedding in paraffin
3. Staining
4. Slicing

89.  2. Embedding in paraffin