HISTOPATHOLOGY

3 X 2 cm

3-5 mm thick

Specimen size for processing

1mm3

For Electron Microscopy

2\.5 x 4 cm

5mm / depth

tissue casette

FIXATION

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pH:

Traditionally:

Autotech:

Em and Histochem:

Rapid Fixation:

For tissues with TB:

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6 - 8

Room Temp

40degC

0-4degC

60degC

100degC

Thickness

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EM:

LM:

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Tissues should not be

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Edematous Lung tissue

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1-2mm2

2cm2

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> 0.4 cm / 4 or 5mm thick EXCEPT

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1-2cm thick

Osmolality

Slightly Hypertonic Solution (400-450 mOsm)

Concentration

10% Formalin

3% Glutaraldehyde

0\.25% Glutaraldehyde (Immunoelectron Microscopy)

Time Duration:

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Primary fixation in buffered formalin:

EM:

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2-6 hrs

3 hrs

Rate of penetration

1mm/hr

Formalin

12-24 hrs

Prolonged storage of 10% formalin

Paraformaldehyde (white precipitate)

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Remedy: add 10% methanol

Brown or Black Crystalline Precipitates

Formic acid with blood

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Remedy: (removal of formalin pigments)

Kardasewitsch’s Method: (70% ethanol & 28% ammonia water)

Lillie’s method: (Hydrogen peroxide & 28% Ammonia water)

Picric Acid Method (Saturated Alcoholic Picric Acid)

1% KOH in 80% Alcohol

Acetone fixatives

\-5 to 4degC

Microwave technique

45-55degC

Penetrate tissue with thickness of 10-15mm

Nitric acid imparts a yellow color due to formation of nitrous acid

Remedy: Add 5% Na Thiosulfate, add urea crystals in conc. soln

Chelating agents

combined with Calcium salts

for immunohistochemical or enzyme staining and electron microscopy

Chelating agents duration

Small specimen: 1-3 weeks

Dense bones: 6-8 weeks or longer

Chelating agents pH:

7-7.4 pH

Post-decalcification:

decalcified tissues are neutralized by

1\.) Immersing in saturated Li2CO3 or 5-10% NaHCO3

2\.) Rinsing in running tap water

3\.) Storing in formol saline containing 15% sucrose or PBS with 15-20% sucrose at 4degC

Ethanol

Boiling point: 78.3degC

BEST dehydrating agent

fast acting

Butanol

Boiling point: 117.7degC

plant and animal microtechniques

odorous

Tertiary Butanol

Boiling point: 82.8degC

Universal Solvent

Pentanol

Boiling point: 128degC

dissolves paraffin

miscible with 90% alcohol, toluene, xylene

Isopropanol

Boiling point: 82.3degC

SUBSTITUTE for ETHANOL

BEST CLEARING AGENT for microwave technique

Paraffin’s melting point for routine work

56degC

Wax bath thermostat:

3degC higher than melting point of wax

Filter in a wax oven

2degC higher than the melting point

Vacuum embedding

negative atmospheric pressure (400-500mmHg)

Heat wax to 100-105degC

water in the wax must be removed by water

10-20% beeswax or ceresin

these are mixed to paraffin wax to give extra hardness

Paraplast

MP: 56-57degC

Mixture of paraffin and Synthetic Plastic Polymer (DMSO)

Embeddol

MP: 56-58degC

similar to paraplast

Bioloid

semi-synthetic; embedding for eyes

Tissue Mat

contains RUBBER

Ester Wax

MP: 46-48degC

water insoluble 95% ethanol soluble

Sliding or Sledge Type Microtome

Water Soluble Waxes

Polyethylene glycols

most common: CARBOWAX

38-42degC / 45-56degC

no need for CLEARING and DEHYDRATION

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PEARSE

* diethyl glycol
* distilled water
* 40% formalin

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BLANK AND MCCARTHY

* 0.02% gelatin and 0.02 potassium dichromate

Wet celloidin

bones, brain sections, teeth

70% alcohol for storage

Dry celloidin

whole eye section

Gilson’s mixture for storage (chloroform and cedarwood oil)

Nitrocellulose method

(Low Viscosity Nitrocellulose)

equal conc. of alcohol and ether

harder tissue block and thinner sections

add plasticizer to prevent cracking

* castor oil
* oleum ricini

< 2-3mm

tissue processing of gelatin

1% phenol

added to prevent molds in gelatin

Embedding for EM

allow tissue sectioning of 80nm of thickness

High Resolution Light Microscopy

renal biopsies: 2um

hematopoietic tissues

epoxy

widest application (EM and LM)

30-40nm

bisphenol A

most stable

cyclohexene dioxide (spurr)

infiltrates FASTEST

highly toxic

carcinogenic

Rocking microtome

Paldwell Trefall

large PARAFFIN embedded blocks

can cut 10-12um

Rotary microtome

Minot

routinely/commonly used

cutting of PARAFFIN embedded tissues

Sliding microtome

Adams

CELLOIDIN sections and hard rough tissue blocks

* base sledge/sledge type - preferred
* standard sliding

Freezing microtome

Queckett

CO2 used as freezing agent

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Cryostat or cold microtome

* rotary microtome inside
* rush frozen section
* -5 to -30 degC (Ave: -20 degC)
* soft tissue paper moistened with ALCOHOL: used to clean knife

Ultrathin microtome

for Electron Miscroscopy

0\.5um

Diamond knife

Vibratome

for unfixed tissues

Plane concave

25mm “shortest”

for base sledge, rotary or rocking microtome

Less concave side

ceLLoidin

More concave side

paraffin

Biconcave knife

120mm “longest”

both sides are concave

cutting PARAFFIN section

rotary microtome

Plane wedge

100mm

FROZEN sections or very hard tissues

Base sledge / sliding microtome

Disposable blades

coated with polytetrafluoroethylene

Glass and Diamond knives

for ULTRATHIN microtome

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* Glass knives - Ralph knives

bevel

cutting facet

found on the TAPERED EDGE OF THE KNIFE

bevel angle

angle formed BETWEEN THE CUTTING EDGE

27-32deg

* knife back / slide on back - maintains the bevel angle

Cutting angle

15deg

maximum penetration of tissue and less distortion

clearance angle

0-15deg

angle formed BETWEEN THE SURFACE OF THE BLOCK and the CUTTING EDGE of the knife

rake angle

angle of the upper surface of facet and surface of tissue block

wedge angle

angle between the sides of knife

Honning

* 10-20 strokes
* heel to toe
* 8x3 inch

Types of hones

* belgium yellow - best result
* arkansas
* fine carborundum - badly nicked knives
* plate glass (8 x 3 x 1 in) - excellent substitute for stone

stropping

3-4 x 18 inches

40 - 120 double strokes

Paraffin section

4-6um sections

celloidin section

10-15um

renal biopsy

2um

ultrathin section

80nm (silver or straw colored sections)

frozen section

4 or 10 -15um (rotary)

tissue block of cold knife

3-5 mm

dew line

point in which sections may be cut at 10um

tissue block of mounting

2-4mm

can be cut from 5-10um

brittle; hard tissue

overdehydration, over-clearing

clearing agent becomes milky

incomplete dehydration

soft and mushy tissue

under-fixation

tissue becomes opaque

under clearing

very hard tissues; tissue shrinkage

overdehydration; overheated of paraffin

air holes on trimmed tissue block

contaminated wax

moist block that tends to crumble

problems in infiltration & embedding

tissue block smells like xylene

under infiltration

Flotation water bath

MP: 10degC below the MP of wax

* overheating - “parched earth”
* enamel black
* Diameter: 11 inch
* height: 4 inch
* 2L capacity
* filled within 1/2 - 1 cm

drying oven

5degC higher than MP of wax

* forceps and squirrel hair brush
* clean slides: 76 x 25mm; 1 - 1.2mm thick; **frosted** (labelled with pencil); **non-frosted** (labelled with diamond pencil/glass etcher)

fishing out

slide with ribbon is stood to drain at the angle of 60-85deg for 2-5 mins

dry the section

* hot plate: 45-55degC for 30-45 mins
* incubator overnight - BEST FOR NERVOUS TISSUE
* wax oven: 56-60degC for 2 hrs
* blower: 50-55degC for 20-30 mins
* bunsen burner until the wax melts - urgent method

freeze drying

* 2mm tissue thickness
* preservation by rapid freezing (quenching) at -160degC
* dessication by transferring the frozen tissue in a vacuum at -40degC

aluminum hematoxylin

* harris hematoxylin
* ehrlich’s hematoxylin
* delafield’s hematoxylin
* mayer’s hematoxylin
* cole’s hematoxylin
* carazzi’s hematoxylin

harris hematoxylin

REGRESSIVE

rippened with MERCURIC OXIDE

staining of SEX CHROMOSOMES

addition of GLACIAL ACETIC ACID - precise nuclear staiing

ehrlich’s hematoxylin

REGRESSIVE

mucin, bone, and cartilage

GLYCERIN is added - slow oxidation and prolong shelf life of hematoxylin

delafield’s hematoxylin

NATURAL rippening

similar longevity to ehrlich’s

mayer’s hematoxylin

REGRESSIVE

rippened with SODIUM IODATE

cole’s hematoxylin

ARTIFICIALLY rippened with ALCOHOLIC IODINE

carazzi’s hematoxylin

ARTIFICIALLY ripened with POTASSIUM IODIDE

Iron hematoxylin

* weigert’s hematoxylin
* heidenhain’s hematoxylin
* loyez hematoxylin
* verhoeff hematoxylin

weigert’s hematoxylin

Mordant/oxidizer: FERRIC AMMONIUM CHLORIDE

for muscle fibers and CT

Heidenhains hematoxylin

Mordant: IRON ALUM

for nuclear and cytoplasmic inclusions

(gray-black)

Loyez hematoxylin

for frozen sections