Staining

Ma. Minda Luz Meneses-Manuguid, M.D.

 Staining
• the process of applying stains/dyes to tissue
sections
• purpose: to make tissues/cells/structures
visible & appreciable; enables one to study
morphology & placement of cells & tissues
• stains/dyes : colored substances:
natural/synthetic;
• different tissues have different affinities for
different dyes
 Pre-staining considerations
• Precautions & proper technique
• Type of tissue
• Component of interest
• Prior reagent used:
 Deparaffinization (sections to water)
 Collodionization / parcelloiden / celloidin
 Frozen sections
 Decalcifying agents
 Adhesives
• Mayer’s egg albumin
• Dried albumin
• Gelatin
• Starch paste
• Plasma celloiden
• Chrome alum adhesive
 Staining
• Histological staining: tissue constituents interact
directly with staining solutions; to demonstrate
general relationship of tissues & cells and
differentiate nucleus from cytoplasm
• Histochemical staining: dyes react chemically with
specific parts of the cell
• Immunohistochemical staining: use of antibodies to
induce a reaction with specific antigenic components
of cells/tissues
 Types of Dye
• Natural dyes
• Synthetic dyes - chromophore
Affinity
 Processes involved
• Osmosis
• Solvation
• Adsorption
• Absorption
 Staining Methods
• Direct • Microanatomical
• indirect • Histochemical
• Progressive • Histological
• Regressive • Vital
• Metachromatic • Intravital
• Differential / • Supravital
• Counterstaining • Metallic impregnation
 Methods of Staining
• Direct – use of aqueous or alcoholic dye sol’n
• Indirect – action of a dye is intensified by adding
another agent (mordant)
• Progressive – tissue elements are stained in sequence;
depends on affinity of tissues to dyes
• Regressive – the tissue is overstained to obliterate
cellular details and then washed & decolorized
• Differential – use of a primary dye (usually basic) then
a differentiating agent (acidic dye) to provide
contrasting colors
 Staining Methods
• Metachromatic – use of specific dyes which
differentiate particular substances by staining them
with a color different from that of the stain itself.
Used to stain Cartilage, mucin, mast cell granules,
amyloid, connective tissues.
• Basic dyes of the thizine & triphenylmethane grps
 Methyl violet/crystal violet ▪ Methylene blue
 Cresyl blue ▪ Toluidine blue
 Safranin ▪ Thionine
 Bismarck brown ▪ Azure A, B, C
 Basic fuchsin
 Staining Methods
• Counterstaining – application of a different color or
stain to provide contrast/background to the structure
being demonstrated
 Cytoplasmic stains: red – EosinY, EosinB, PhloxineB; yellow –
Picric acid, OrangeG, rose Bengal; green – light greenSF,
Lissamine green
 Nuclear stains: red – neutral Red, Safranin O, Carmine,
Hematoxylin; blue – Methylene blue, Toluidine blue, Celestine
blue
• Metallic impregnation – certain tissue elements
reduce colorless metallic salt solutions to produce a
black (usually) precipitate on the surface of the tissue
 Silver nitrate ▪ Gold chloride
 Mordants & Accentuators
• Mordants: combine with dye to form a colored
“lake” which then combines with the tissue, rendering
it insoluble in ordinary aqueous & alcoholic solvents
– easily counterstained
 Potassium alum (in Ehrlich’s hematoxylin)
 Iron (in Weigert’s hematoxylin)
• Accentuator – accelerates rate of staining by
increasing staining power & selectivity of the dye
 Potassium hydroxide in Loeffler’s methylene blue
 Phenol in carbol fuchsin
 Vital Staining
• selective staining of living cell constituents;
cytoplasmic phagocytosis of the dye results in
coloring of cytoplasmic structures (the nucleus is
resistant to entry of the dye)
• Intravital staining – injection of the dye into any part
of the body
• Supravital staining – staining of living cells
immediately after removal from the living body
• commonly used dyes in vital staining:
 Neutral red ▪ Nile blue
 Janus green ▪ Thionine
 Tryphan blue ▪ Toluidine blue
 Hematoxylin & Eosin
• routine staining method
• differential, regressive staining – makes use of a
combination of dyes sequentially used to render
contrasting colors to acidophilic & basophilic
constituents
• results:
 Nuclei – blue to blue-black ▪ cartilage – pink-blue-dk blue
 Karyosome – dark blue ▪ Calcium, Bone – purple-blue
 Cytoplasm, Proteins – pale pink ▪ decal Bone, Collagen,
 RBCs, eosinophil granules – Osteoid – pink
bright red to orange-red ▪ muscle fibers – deep pink
 Keratin – dark pink ▪ plasma cells – purple pink
 Results
• collagen............................pale pink
• muscle...............................deep pink
• acidophilic cytoplasm.......red
• basophilic cytoplasm.........purple
• nuclei..................................blue
• erythrocytes........................cherry red
 Bluing reagents for H & E
• 0.1% Sodium Bicarbonate:
 Sodium bicarbonate 1 g + Distilled water 1000 ml    
 Mix to dissolve. The pH will be around 8.0 Store this soln at room temp.
 Bluing for 30 sec to 1 min after hematoxylin staining and clearing/differentiation.
Note: this sol’n worked better than ammonia water sol’n.
• 0.2% Ammonia Water Solution (Bluing):
 Ammonium hydroxide (conc) 2 ml + Distilled water 1000 ml
 Mix well. The pH will be around 10.0 Store this soln at room temp.
 Bluing for 30 sec to 1 min after hematoxylin staining and clearing/differentiation. 
Note: this soln is not as good as sodium bicarbonate.
• Lithium Carbonate Solution (Saturated):
 Lithium carbonate 1.54 g + Distilled water 100 ml
 Mix to dissolve and store at room temp.
 Bluing for 30 sec to 1 min after hematoxylin staining and clearing/differentiation.
Clearing (Differentiation) Rgt
for H&E Staining
• 1% Acid Alcohol Solution:
 Hydrochloric acid ---------------- 10 ml
 70% ethanol ---------------------- 1000 ml
 Mix well and store at room temperature.
 Differentiate 30 second to 2 minutes after hematoxylin
over-stain.
 50 um paraffin sections require 2 minutes differentiation.
 H & E staining method
1. Clearing – xylene bath – 2nd xylene bath
2. Absolute Ethanol – 95% Ethanol
3. Rinse – running water
4. Hematoxylin
5. Wash – running water
6. Differentiate – 1% acid Alcohol
7. Rinse – tap water
8. Blueing – ammonia water
9. Wash – running water
10. Counterstain – Eosin
11. Wash & Differentiate
12. Dehydrate
13. Clear & Mount
 H&E Staining Protocol -
Harris
Solutions and Reagents:  
• 1% Acid Alcohol Solution (for differentiation):
 Hydrochloric acid 1 ml + 70% ethanol 100 ml
 Mix well. 
• 0.2% Ammonia Water Solution (Bluing):
 Ammonium hydroxide (conc) 2 ml + Distilled water 1000 ml
 Mix well 
• Lithium Carbonate Solution (Saturated):
 Lithium carbonate 1.54 g + Distilled water 100 ml
 Mix well.
• Eosin-Phloxine B Solution:
• Eosin Stock Solution:
 Eosin Y 1 g + Distilled water 100 ml
 Mix to dissolve.
• Phloxine Stock Solution:
 Phloxine B 1 g + Distilled water 100 ml
 Mix to dissolve.
• Eosin-Phloxine B Working Solution:
 Eosin stock solution 100 ml
 Phloxine stock solution 10 ml
 Ethanol (95%) 780 ml
 Glacial acetic acid 4 ml
 Mix well.
Alternate for Eosin-Phloxine B
Solution
Eosin Y Solution:
• Eosin Y Stock Solution (1%):
 Eosin Y --------------------------------------- 10 g
 Distilled water ------------------------------- 200 ml
 95% Ethanol ---------------------------------- 800 ml
 Mix to dissolve and store at room temperature.
• Eosin Y Working Solution (0.25%):
 Eosin Y stock solution ------------------ 250 ml
 80% Ethanol ------------------------------ 750 ml
 Glacial acetic acid (concentrated) ----- 5 ml
 Mix well and store at room temperature.
 Harris Hematoxylin
• Hematoxylin Solution (Harris):
 Potassium or ammonium (alum) ----------- 100 g
 Distilled water ------------------------------- 1000 ml
 Heat to dissolve.
 Add 50 ml of 10% alcoholic hematoxylin solution and heat to boil
for 1 minute.
 Remove from heat and slowly add 2.5 g of mercuric oxide (red).
 Heat to the solution and until it becomes dark purple color.
 Cool the solution in cold water bath and add 20 ml of glacial
acetic acid (concentrated).
 Filter before use.
 Procedure
1.  Deparaffinize sections, 2 changes of xylene, 10 minutes each.
2.  Re-hydrate in 2 changes of absolute alcohol, 5 minutes each.
3.  95% alcohol for 2 minutes and 70% alcohol for 2 miuntes.
4.  Wash briefly in distilled water.
5.  Stain in Harris hematoxylin solution for 8 minutes.
6.  Wash in running tap water for 5 minutes.
7.  Differentiate in 1% acid alcohol for 30 seconds.
8.  Wash running tap water for 1 minute.
9.  Bluing in 0.2% ammonia water or saturated lithium carbonate soln for
30 sec to 1 min
10. Wash in running tap water for 5 minutes.
11. Rinse in 95% alcohol, 10 dips.
12. Counterstain in eosin-phloxine B soln/eosin Y soln for 30 sec to 1 min
13. Dehydrate through 95% alcohol, 2 changes of absolute alcohol, 5 min
each.
14. Clear in 2 changes of xylene, 5 minutes each.
15. Mount with xylene based mounting medium.
• Results:
• Nuclei ---- blue
• Cytoplasm ----- pink to red
 H & E Protocol – Mayer’s
• Hematoxylin Solution (Mayer):
 Potassium or ammonium (alum) -------- 50 g
 Hematoxylin ------------------------------- 1 g
 Sodium iodate ----------------------------- 0.2 g
 Citric acid ---------------------------------- 1 g
 Distilled water ----------------------------- 1000 ml
• Stir to dissolve the chemicals in the order listed
above. For example, dissolve alum in 1000 ml
distilled water first. When alum is completely
dissolved, add hematoxylin. When hematoxylin is
completely dissolved, add sodium iodate, etc. 
 Procedure
1. Deparaffinize sections, 2 changes of xylene, 10 minutes each.
2. Re-hydrate in 2 changes of absolute alcohol, 5 minutes each.
3. 95% alcohol for 2 minutes and 70% alcohol for 2 miuntes.
4. Wash briefly in distilled water.
5. Stain in Mayer hematoxylin solution for 8 minutes.
6. Wash in warm running tap water for 10 minutes.
7. Rinse in distilled water.
8. Rinse in 95% alcohol, 10 dips.
9. Counterstain in eosin-phloxine B solution (or eosin Y solution) for
30 seconds to 1 minute.
10. Dehydrate through 95% alcohol, 2 changes of absolute alcohol, 5
minutes each.
11. Clear in 2 changes of xylene, 5 minutes each.
12. Mount with xylene based mounting medium.
• Results: 
• Nuclei ------ blue
• Cytoplasm ---- pink to red
Hematoxylin and Eosin (H&E)
Staining Protocol
 
• Principle: The oxidation product of hematoxylin is hematin, and is the
active ingredient in the staining solution.  Hematoxylin is not classified
as a dye since the molecule possesses no chromophore.  The in
situ oxidation of hematoxylin is effected by the addition of a strong
oxidant to the stain, in this case sodium iodate.  Lillie’s variant of
Mayer’s hemalum is discussed in Lynch et al. (pp 1032)
•  Hematin exhibits indicator-like properties, being blue and less soluble
in aqueous alkaline conditions, and red and more soluble in alcoholic
acidic conditions.  In acidic conditions, hematin binds to lysine
residues of nuclear histones by linkage via a metallic ion mordant, in
this case aluminium.  To ensure saturation of chemical binding sites,
the stain is applied longer than necessary, resulting in the overstaining
of the tissues with much non-specific background coloration.  This
undesirable coloration is selectively removed by controlled leaching in
an alcoholic acidic solution, (acid alcohol), the process being termed
"differentiation".  Differentiation is arrested by returning to an
alkaline environment, whereupon the hematin takes on a blue hue, the
process of "blueing-up".  The hematin demonstrates cell nuclei.
 H & E Staining Protocol
• Full cellular detail is obtained by counterstaining with the
eosin mixture.  There are three commonly used forms of
eosin - eosin Yellowish (tetrabromofluorescein, disodium
salt CI 45380), eosin Bluish (the dinitro- dibromo-
derivative CI 45400), and eosin Alcohol Soluble (the ethyl
derivative CI 45386), the former is preferred.  Color
enhancement is achieved by fortifying the stain with
phloxine, a chemical member of the same family as
eosin( halogenated fluorosceins).  The mechanism of their
staining is not fully understood, but is believed to be of an
electrostatic nature.  Visualisations most acceptable to the
histologist are obtained by applying the dyes in acidic
conditions, whereby more intense specific colourations are
obtained, the more acidic tissue components taking up the
dye to a greater intensity, hence the addition of acetic acid.
 H & E Staining Protocol
• Technical Points
1. (step 2) - The length of time necessary to over-stain the tissues will depend
upon fixation and the type of alum haematoxylin employed. (Lillie Mayer's alum
haematoxylin-formalin fixed tissues 5 mins).

Tissue Type  Haematoxylin Acid alcohol Eosin Comment

0.3%
Routine tissues  4 min See technical 2 min
point 2
Renal biopsies  10 min 1-2 sec 2-4 min Check
staining
Decals 10 min 1-2 sec 30 sec Check
staining after
blueing. Hx
step may
need to be
repeated if
prolonged decal.
 H & E Staining Protocol
2. (step 4) - Differentiation with acid alcohol requires some practical
experience to ascertain the correct end-point, since the acid solution
alters the colour of the tissue to red.  The correct end-point is when,
after blueing up, the background is almost colourless.  For renal
biopsy sections, two quick dips in 0.3% acid alcohol are all that is
required
3.  (step 6) - If Scott's tap water substitute is employed, blueing up is
achieved in a much shorter time.
4. (step 8) - Eosin is highly soluble in water.  Over-staining is removed by
washing in running water.
5.  Fixation - Not critical.  Acidic fixatives will give a more eosinophilic
result.  Picric acid containing fixatives give an overall enhanced
result.  Acidic decalcifying fluids give poor nuclear staining.
6.  Renals - 10% buffered formalin.  Sections cut at 2m
 H & E Staining Protocol
• Method 
1. Bring sections to distilled water
2. Stain nuclei with the alum haematoxylin (see note)
3. Rinse in running tap water
4. Differentiate with 0.3% acid alcohol (see note)
5. Rinse in running tap water
6. Rinse in Scott's tap water substitute (see note)
7. Rinse in tap water
8. Stain with eosin   2 mins
9. Dehydrate, clear and mount.
 Re-staining
• Removal of coverslip: 24 hrs xylene or
heating
• Xylene 30 mins
• KMnO4 5 – 10 min
• Oxalic acid 5 mins
• Repeat washing
Mounting
 Mounting Media
• Aqueous media
 Water with glycerine & jelly
 Glycerin
 Gum arabic / Farrant’s medium
 Karo corn syrup
• Resinous media
 Canada balsam
 DPX
 Xam
 Permount
 Clarite
 H&E
• Nuclei – blue with some metachromasia
• Cytoplasm – various shades of pink
identifying different tissue components
Thank You !