VON KOSSA'S METHOD CALCIUM

PURPOSE: Abnormal deposits of calcium may be found in any area of the

body. With the H&E stain, calcium appeared deep blue-purple.
PRINCIPLE: Tissue sections are treated with silver nitrate solution. The
calcium is reduced by the strong light and replaced with silver deposits,
visualized as metallic silver.
CONTROL: Tissue
undecalcified bone.

containing

known

positive

calcium

deposits,

or

FIXATIVE: 10% formalin

TECHNIQUE: Cut paraffin sections at 4.
EQUIPMENT: Acid cleaned glassware, 60-watt lamp, foil or mirror.
REAGENTS:

5% Silver Nitrate Solution: Silver nitrate 25.0 gm

Distilled water 500.0 ml

Mix well, pour into acid clean brown bottle. Store in the refrigerator. Solution
is stable for 1 year.
CAUTION: Avoid contact and inhalation.

5% Hypo

CAUTION: Avoid contact and inhalation.

Nuclear Fast Red

SAFETY:
-Wear gloves, goggles and lab coat. Avoid contact and inhalation.
-Silver nitrate; Severe skin and eye irritant. Ingestion will produce violent GI
distress.
Tumorigenic. Oxidizer.

-Hypo (sodium thiosulfate); Powder is an eye, skin and respiratory irritant.

Ingestion may cause GI distress.

MINERALS & PIGMENTS VON KOSSA'S METHOD CALCIUM

PROCEDURE:
1. Deparaffinize and hydrate to distilled water.
2. 5% Silver solution, place in bright sunlight, or in front of a 60-watt lamp,
place foil (or mirror) behind the jar to reflect the light. Leave for 1 hour or
until calcium turns black.
3. Rinse in distilled water, 3 changes.
4. 5% Hypo, 5 minutes.
5. Wash in tap water, rinse in distilled.
6. Nuclear-fast Red, 5 minutes.
7. Wash in water.
8. Dehydrate, clear, and coverslip.

RESULTS:
Calcium salts: black
Nuclei: red
Cytoplasm: pink

FROZEN TISSUE STAINING

Preparation of Frozen Sections for Sectioning
Materials neeed:

2-methylbutane (isopentane)

Liquid Nitrogen

Dry ice

Peel-Away base molds

Frozen tissue matrix (OCT or Cryomatrix)

Long forceps

Necropsy tools

Superfrost Plus slides

1. Label base mold and partially fill the mold with frozen tissue matrix.
2. Sacrifice animal by prescribed and approved euthanasia techniques.
3. Remove desired tissues, trim and cut tissue no more than 5 mm thick.
Place in pre-labeled base molds filled with frozen tissue matrix.
Arrange tissue in the matrix near the bottom so tissue is easily
exposed when sections are cut.
4. Place a stainless steel beaker of 2-methylbutane in liquid nitrogen and
allow to cool adequately. Place base mold with tissue into the beaker of
cold 2-methylbutane and quickly immerse the block. Allow the tissue
matrix to solidify completely and remove block from 2-methylbutane

and place on dry ice or in the -20C cryostat. NOTE: If block is left in 2methylbutane too long, the block may crack.
5. Store blocks in the -80C freezer until ready for sectioning.

Sectioning of Frozen Tissues

1. Before cutting sections, allow the temperature of the block to
equilibrate to the temperature of the cryostat (typically -20C).
2. Place the tissue block on the cryostat specimen disk. Adjust the
positioning of the block to align the block with the knife blade. Cut
tissue block until the desired tissue is exposed.
3. Cut sections of the desired thickness (usually 5 m), place the sections
on a Fisher Superfrost slide and dry overnight at room temperature
(RT).
4. Fix slides by immersion in cold acetone (-20C) for 2 minutes or other
suitable fixative (e.g. alcohol, formal alcohol, formalin, etc.), air dry at
RT and proceed to staining.
5. Alternatively, the frozen section slides can be stored for a short period
of time at -70C in a sealed slide box. When ready to stain, remove
slides from freezer and warm to -20C in the cryostat or -20C freezer,
fix for 2 minutes in cold fixative (acetone or other suitable fixative) and
allow to come to RT to continue with the staining.

Standard Immonuhistochemical Staining Procedure for Frozen

Sections
Please

read

entire

procedure

before

staining

sections.

Perform

all

incubations in a humid chamber and do not allow sections to dry out.

Isotype and system controls should also be run and must be matched to the
isotype of each primary antibody to be tested.
Materials needed:

Phosphate Buffered Saline (PBS) (cat. no. 554781)

H2O2 Solution

Antibody Diluent for IHC (Cat. No. 559148)

Streptravidin-Horseradish Peroxidase (Cat. No. 550946)

DAB Substrate Kit (Cat. No. 550880)

Hematoxylin

Bluing Reagent

Graded alcohols

Xylene

Procedure:
1. Label slides with a solvent resistant pen and demarcate the tissue if
required.
2. Rinse slides 3x in PBS, to remove the tissue-freezing matrix.
3. Block endogenous peroxidase activity by incubating the slides in 0.3%
H2O2 solution in PBS for 10 minutes.
4. Rinse slides 3x in PBS, 2 minutes each time.
5. Block non-specific binding by incubating with blocking buffer (10%
serum from host species of secondary antibody diluted in PBS or 10%
FBS in PBS) for 30-60 min at RT in a humidified chamber.
6. Dilute the primary antibody in the Antibody Diluent for IHC.
Alternatively, a buffered solution with a source of protein can be used
as antibody diluent. Apply the diluted antibody to the tissue sections
on the slide. Incubate for 1 hour at RT in a humidified chamber.
7. Rinse slides 3x in PBS, 2 minutes each time.
8. Dilute the biotinylated secondary antibody in the Antibody Diluent for
IHC. Alternatively, a buffered solution with a source of protein can be
used as antibody diluent. Apply to the tissue sections on the slide and
incubate for 30 minutes at RT.

9. Rinse slides 3x in PBS, 2 minutes each time.

10.
Apply the Streptravidin-Horseradish Peroxidase pre-diluted to the
tissue sections on the slide and incubate for 30 minutes at RT.
11.

Rinse slides 3x in PBS, 2 minutes each time.

12.
Prepare DAB substrate solution by adding 1 drop of DAB
chromagen to every 1 ml of DAB buffer. (When using other substrates
follow manufacturer's recommendations.)
13.
SAFETY NOTE: DAB is a suspect carcinogen. Handle with care.
Wear gloves, lab coat and eye protection.
14.
Drain PBS from slides and apply the DAB substrate solution.
Allow slides to incubate for 5 minutes or until the desired color
intensity is reached.
15.

Wash 3X in water, 2 minutes each time.

16.

Counterstain slides:
a. Dip twice in Hematoxylin.
b. Rinse thoroughly in water.
c. Dip twice in Bluing Reagent or dilute ammonia water.
d. Rinse thoroughly in water.

17.
Dehydrate through 4 changes of alcohol (95%, 95%, 100% and
100%). Clear in 3 changes of xylene (or xylene substitute) and
coverslip using mounting solution.

Papanicolaou (PAP) Staining

Introduction
Papanicolaou stain (also Papanicolaous stain or PAP stain) is the most important stain utilized in the
practice of Cytopathology. It is a polychromatic stain containing multiple dyes to differentially stain
various components of the cells. This technique was developed by George Papanicolaou, the father
of Cytopathology. This method is used to differentiate cells in the smear preparation of various

gynecological specimens (pap smears), materials containing exfoliative cells and material from fine
needle aspiration.

Objectives of Papanicolaou Stain

Papanicolaou described three chief objectives for staining of cytological
smears:

Definition of nuclear details : Because of the widespread muclear

abnormalities of cancer cells and their diagnostic significance, good
staining of the nucleus is of primary importance.

Transparency of cytoplasm : This is of particular importance

because of the varying thickness and the frequent overlapping of cells.

Differentiation of cells : Differences in the staining reaction such as

that between acidophilic and basophilic cells help greatly in the
identification of certain cell types found in smears.

Principle of Papanicolaou Stain

Papanicolaou stain includes both acidic and basic dyes. Acidic dye stains the
basic components of the cell and basic dye stain the acidic components of
the cell. The polychromatic PAP stain involves five dyes in three solutions.
Hematoxylin : Natural dye hematoxylin is the nuclear stain which stains cell
nuclei blue. It has affinity for chromatin, attaching to sulphate groups on the
D.N.A. molecule. Harris hematoxylin is the commonest cytologically
although Gills hematoxylin and Hematoxylin S can be used.
Orange Green 6 : This is the first acidic counterstain (cytoplasmic stain)
which stains matured and keratinized cells. The target structures are staine d
orange in different intensities.
Eosin Azure : This is the second counterstain which is a polychrome mixture
of eosin Y, light green SF and Bismarck brown.

Eosin Y gives a pink colour to cytoplasm of mature squamous cells, nucleoli,

cilia and red blood cells. Staining solutions commonly used in cytology are EA
31 and EA 50, while EA 65
Light green SF stains blue to cytoplasm of metabolically active cells like
parabasal squamous cells, intermediate squamous cells and columnar cells.
Bismarck brown Y stains nothing and sometimes it is often omitted.
Composition and Preparation of Reagents
1. Harris hematoxylin :
Hematoxylin = 5g
Ethanol = 50ml
Potassium alum = 100g
Distilled water (50C) = 1000ml
Mercuric oxide = 2-5g
Glacial acetic acid = 40ml
2. Orange G 6 :
Orange G (10% aqueous) = 50ml
Alcohol = 950ml
Phosphotungstic acid = 0-15g
3. EA 50 :
0.04 M light green SF = 10ml
0.3M eosin Y = 20ml
Phosphotungstic acid = 2g
Alcohol = 750ml
Methanol = 250ml
Glacial acetic acid = 20ml
Filter all stains before use.

Procedure of Papanicolaou Staining

Both progressive and regressive nuclear staining techniques can be used
in Papanicolaou stain. Before staining, Wet fixation immediately with
Cytology spray fixative 96% ethanol for minimum 30 min is required.

Procedure of Progressive Papanicolaou Staining Method

In the progressive method, the nucleus is stained with hematoxylin to a
intensity desired. The intensity of the nuclear staining is controlled by the
immersion of the slide into a blueing agent. Most commonly used blueing
agent is Sotts tap water (pH 8.02).
Step

Reagent

Time

1.

95% Alcohol (Fixation)

15-30 minutes

2.

80% Alcohol

2 minutes

3.

60% Alcohol

2 minutes

4.

Distilled Water

5 dips

5.

Distilled Water

5 dips

6.

Hematoxylin stain

3 minutes

7.

Distilled Water

3 minutes

8.

60% Alcohol

2 minutes

9.

80% Alcohol

2 minutes

10.

95% Alcohol

2 minutes

11.

Orange G Stain

3 minutes

12.

95% ALcohol

2 minutes

13.

95% Alcohol

2 minutes

14.

Eosin Azure Stain

3 minutes

15.

95% Alcohol

2 minutes

16.

95% Alcohol

2 minutes

17.

95% Alcohol

2 minutes

18.

95% Alcohol

2 minutes

19.

Absolute Alcohol

2 minutes

20.

Absolute Alcohol

2 minutes

21.

Absolute Alcohol

2 minutes

22.

Absolute Alcohol+Xylene (1:1)

2 minutes

23.

Xylene

2 minutes

24.

Xylene

2 minutes

25.

Xylene

Till clear

26.

Mount in D.P.X

Procedure of Regressive Papanicolaou Staining Method

When using the regressive staining method, the nucleus is deliberately overstained with a non-acidified haematoxylin. The excess stain is removed with
dilute hydrochloric acid solution (acid water). The decolourising process is
then stopped by immersing the slide in running tap water. Timing is crucial in
the regressive method as de-staining may lead to a hyperchromatic nucleus
becoming hypochromatic.
Step

Reagent

Time

1.

90% Alcohol (Fixation)

15-30 minutes

2.

80% Alcohol

2 minutes

3.

60% Alcohol

2 minutes

4.

Distilled Water

5 dips

5.

Distilled Water

5 dips

6.

Hematoxylin stain

3 minutes

7.

Distilled Water

10 seconds

8.

1% Acid Alcohol

10 seconds (1 dip)

9.

Distilled Water

10 seconds

10.

Scotts Tap Water

2-3 minutes

11.

Running Tap Water

2 minutes

12.

60% Alcohol

2 minutes

13.

80% Alcohol

2 minutes

14.

95% Alcohol

2 minutes

15.

Orange G Stain

3 minutes

16.

95% ALcohol

2 minutes

17.

95% Alcohol

2 minutes

18.

Eosin Azure Stain

3 minutes

19.

95% Alcohol

2 minutes

20.

95% Alcohol

2 minutes

21.

95% Alcohol

2 minutes

22.

95% Alcohol

2 minutes

23.

Absolute Alcohol

2 minutes

24.

Absolute Alcohol

2 minutes

25.

Absolute Alcohol

2 minutes

26.

Absolute Alcohol+Xylene (1:1)

2 minutes

27.

Xylene

2 minutes

28.

Xylene

2 minutes

29.

Xylene

Till clear

30.

Mount in D.P.X

Results and Interpretation of Papanicolaou Staining

Nuclei : Blue
Acidophilic cells : Red
Basophilic cells : Blue Green
Erythrocytes : Orange-red
Keratin : Orange-red
Superficial cells : Pink
Intermediate and Parabasal Cells : Blue Green
Eosinophil : Orange Red
Candida : Red
Trichomonas : Grey green