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
