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• indirect• Progressive• Regressive• Metachromatic• Differential /• Counterstaining

• Microanatomical• Histochemical• Histological• Vital • Intravital• Supravital • 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 method1. Clearing – xylene bath – 2nd xylene

bath2. Absolute Ethanol – 95% Ethanol 3. Rinse – running water4. Hematoxylin5. Wash – running water6. Differentiate – 1% acid Alcohol7. Rinse – tap water8. Blueing – ammonia water9. Wash – running water10. Counterstain – Eosin11. Wash & Differentiate12. Dehydrate13. 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 min10. 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 min13. 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 2Renal 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 Protocol2. (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 water2. Stain nuclei with the alum haematoxylin

(see note)3. Rinse in running tap water4. Differentiate with 0.3% acid alcohol (see

note)5. Rinse in running tap water6. Rinse in Scott's tap water substitute (see

note)7. Rinse in tap water8. Stain with eosin   2 mins9. 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 !