continuation of part two

read tissue processing part one here 

Tissue Processing : Factors, Steps Of Tissue Processing, Types -part 1

3.  Embedding

  • It is the process by which tissues are surrounded by a medium such as agar, gelatine, or wax which when solidified will provide sufficient external support during sectioning.

Image result for tissue processingEmbedding

Properties of embedding media

Ideally an infiltrating and embedding medium should be

  • soluble in processing fluids
  • suitable for sectioning and ribboning
  • molten between 30°C and 60°C
  • translucent or transparent; colorless
  • Stable
  • Homogeneous
  • capable of flattening after ribboning
  • non-toxic
  • Odorless
  • easy to handle
  • inexpensive

Paraffin wax

  • most popular embedding medium in histopathology
  • It is a mixture of long chained hydrocarbons produced in the cracking of mineral oil.
  • Paraffin wax permeates the tissue in liquid form and solidifies rapidly when cooled.
  • It has a wide range of melting points ranging from 47 to 64°C which signifies its use in the different climatic
  • Heating the paraffin wax to a high temperature alters the properties of the wax.
  • It is inexpensive and provides quality sections
  • Compatible with most routine and special stains

Paraffin wax additives

  • These additives helps to increase hardness
  • Substances added to paraffin wax include beeswax, rubber, ceresin, plastic polymers and diethylene glycol distearate.
  • Many of these additives had a higher melting point than paraffin wax and make the tissue more brittle.

MODIFIED PARAFFIN WAXES

  • The properties of paraffin wax are improved for histological purposes by the inclusion of substances added alone or in combination to the wax:
  • improve ribboning: prolong heating of paraffin wax at high temperatures or use micro-crystalline wax
  • stearic acid :  increase hardness
  • spermaceti or phenanthrene : decrease melting point
  • 5% ceresin, 0.1-5% beeswax, rubber, asphalt, bayberry wax, or phenanthrene : improve adhesion between specimen and wax (alter crystalline morphology)
  • Piccolyte 115, a thermoplastic terpene resin added at the rate of 5%-10% to the infiltrating wax
  • Plastic polymers such as polyethylene wax, added to improve adhesion, hardness and plasticity
  • Dimethyl sulphoxide (DMSO) added to proprietary blends of plastic polymer paraffin waxes reduces infiltration times and facilitates thin sectioning.

Image result for paraffin blocks


Paraffin Blocks

Alternative embedding media

Resin

  • Resin is used exclusively as the embedding medium for electron microscopy, ultra-thin sectioning for high resolution and also for undecalcified bone.

Agar

  • Agar alone does not provide sufficient support for sectioning tissues.
  • Its main use is as a cohesive agent for small friable pieces of tissue after fixation (double embedding)
  • Fragments of tissue are embedded in melted agar, allowed to solidify and trimmed for routine processing.

Gelatin

  • Gelatin is primarily used in the production of sections of whole organs using the Gough-Wentworth technique and in frozen sectioning.
  • It is rarely used.

Celloidin

  • The use of celloidin or LVN (low viscosity nitrocellulose) is discouraged because of the special requirements needed to house the processing reagents and the limited use these types of sections have in neuropathology.
  • It is rarely used.

Double embedding

  • It is the process by which tissues are first embedded or fully infiltrated with a supporting medium such as agar or nitrocellulose, then infiltrated a second time with wax in which they are also embedded.

Embedding tissues in paraffin wax

Requirements for embedding are as follows:
  • a supply of clean, filtered paraffin wax held at 2-4°C above its melting point.
  • a paraffin dispenser
  • a cold plate to rapidly cool the wax.
  • a supply of moulds in which to embed the tissues.
  • Paraffin wax is dispense automatically from a nozzle into a suitably sized mold.
  • The tissue is oriented in the mold, a cassette is attached.
  • The mold is placed on a small cooling area to allow the paraffin
Overnight processing schedule
Image result for histokinette

Reagents

Time

Temperature 

10% Formalin1 h38°C
10% Formalin1 h38°C
50% Alcohol/formalin1 h38°C
70% Alcohol1 h38°C
95% Alcohol1 h38°C
95% Alcohol40 min38°C
100% Alcohol1 h38°C
100% Alcohol40 min38°C
Xylene1 h38°C
Xylene30 min38°C
Paraffin30 min38°C
Paraffin30 min38°C
Paraffin30 min38°C
Paraffin30 min38°C

Microtome cutting

The tissue block is attached to microtome and ribbons are cut.

Image result for microtomeMicrotome

Equipment required for Paraffin section cutting

  • Flotation (water) bath
  • Slide drying oven or hot plate
  • Fine pointed or curved forceps
  • Sable or camel haired brush
  • Scalpel
  • Slide rack
  • Clean slides
  • Chemical-resistant pencil or pen

Water Bath

  • water bath is used for floating out tissue ribbons after sectioning.
  • The trailing end of the ribbon making contact with the water first
  • The temperature of the water in the bath should be 10°C below the melting point of the paraffin.
  • Alcohol or a small drop of detergent may be added to the water allowing the section to flatten out with greater ease
  • 30 seconds are long enough for a ribbon to flatten

Image result for water bathWater Bath

Slides

  • For normal routine work 76 × 25 mm slides are universally used.
  • Those with thickness of 1.0–1.2 mm are preferred

Section adhesives


  • Albumen
  • gelatin
  • Starch
  • Poly-L-lysine (PLL)
  • 3-aminopropyltriethoxysilane (APES)

tissue processing part 2

continuation of part two

read tissue processing part one here 

Tissue Processing : Factors, Steps Of Tissue Processing, Types -part 1

3.  Embedding

  • It is the process by which tissues are surrounded by a medium such as agar, gelatine, or wax which when solidified will provide sufficient external support during sectioning.

Image result for tissue processingEmbedding

Properties of embedding media

Ideally an infiltrating and embedding medium should be

  • soluble in processing fluids
  • suitable for sectioning and ribboning
  • molten between 30°C and 60°C
  • translucent or transparent; colorless
  • Stable
  • Homogeneous
  • capable of flattening after ribboning
  • non-toxic
  • Odorless
  • easy to handle
  • inexpensive

Paraffin wax

  • most popular embedding medium in histopathology
  • It is a mixture of long chained hydrocarbons produced in the cracking of mineral oil.
  • Paraffin wax permeates the tissue in liquid form and solidifies rapidly when cooled.
  • It has a wide range of melting points ranging from 47 to 64°C which signifies its use in the different climatic
  • Heating the paraffin wax to a high temperature alters the properties of the wax.
  • It is inexpensive and provides quality sections
  • Compatible with most routine and special stains

Paraffin wax additives

  • These additives helps to increase hardness
  • Substances added to paraffin wax include beeswax, rubber, ceresin, plastic polymers and diethylene glycol distearate.
  • Many of these additives had a higher melting point than paraffin wax and make the tissue more brittle.

MODIFIED PARAFFIN WAXES

  • The properties of paraffin wax are improved for histological purposes by the inclusion of substances added alone or in combination to the wax:
  • improve ribboning: prolong heating of paraffin wax at high temperatures or use micro-crystalline wax
  • stearic acid :  increase hardness
  • spermaceti or phenanthrene : decrease melting point
  • 5% ceresin, 0.1-5% beeswax, rubber, asphalt, bayberry wax, or phenanthrene : improve adhesion between specimen and wax (alter crystalline morphology)
  • Piccolyte 115, a thermoplastic terpene resin added at the rate of 5%-10% to the infiltrating wax
  • Plastic polymers such as polyethylene wax, added to improve adhesion, hardness and plasticity
  • Dimethyl sulphoxide (DMSO) added to proprietary blends of plastic polymer paraffin waxes reduces infiltration times and facilitates thin sectioning.

Image result for paraffin blocks


Paraffin Blocks

Alternative embedding media

Resin

  • Resin is used exclusively as the embedding medium for electron microscopy, ultra-thin sectioning for high resolution and also for undecalcified bone.

Agar

  • Agar alone does not provide sufficient support for sectioning tissues.
  • Its main use is as a cohesive agent for small friable pieces of tissue after fixation (double embedding)
  • Fragments of tissue are embedded in melted agar, allowed to solidify and trimmed for routine processing.

Gelatin

  • Gelatin is primarily used in the production of sections of whole organs using the Gough-Wentworth technique and in frozen sectioning.
  • It is rarely used.

Celloidin

  • The use of celloidin or LVN (low viscosity nitrocellulose) is discouraged because of the special requirements needed to house the processing reagents and the limited use these types of sections have in neuropathology.
  • It is rarely used.

Double embedding

  • It is the process by which tissues are first embedded or fully infiltrated with a supporting medium such as agar or nitrocellulose, then infiltrated a second time with wax in which they are also embedded.

Embedding tissues in paraffin wax

Requirements for embedding are as follows:
  • a supply of clean, filtered paraffin wax held at 2-4°C above its melting point.
  • a paraffin dispenser
  • a cold plate to rapidly cool the wax.
  • a supply of moulds in which to embed the tissues.
  • Paraffin wax is dispense automatically from a nozzle into a suitably sized mold.
  • The tissue is oriented in the mold, a cassette is attached.
  • The mold is placed on a small cooling area to allow the paraffin
Overnight processing schedule
Image result for histokinette

Reagents

Time

Temperature 

10% Formalin1 h38°C
10% Formalin1 h38°C
50% Alcohol/formalin1 h38°C
70% Alcohol1 h38°C
95% Alcohol1 h38°C
95% Alcohol40 min38°C
100% Alcohol1 h38°C
100% Alcohol40 min38°C
Xylene1 h38°C
Xylene30 min38°C
Paraffin30 min38°C
Paraffin30 min38°C
Paraffin30 min38°C
Paraffin30 min38°C

Microtome cutting

The tissue block is attached to microtome and ribbons are cut.

Image result for microtomeMicrotome

Equipment required for Paraffin section cutting

  • Flotation (water) bath
  • Slide drying oven or hot plate
  • Fine pointed or curved forceps
  • Sable or camel haired brush
  • Scalpel
  • Slide rack
  • Clean slides
  • Chemical-resistant pencil or pen

Water Bath

  • water bath is used for floating out tissue ribbons after sectioning.
  • The trailing end of the ribbon making contact with the water first
  • The temperature of the water in the bath should be 10°C below the melting point of the paraffin.
  • Alcohol or a small drop of detergent may be added to the water allowing the section to flatten out with greater ease
  • 30 seconds are long enough for a ribbon to flatten

Image result for water bathWater Bath

Slides

  • For normal routine work 76 × 25 mm slides are universally used.
  • Those with thickness of 1.0–1.2 mm are preferred

Section adhesives


  • Albumen
  • gelatin
  • Starch
  • Poly-L-lysine (PLL)
  • 3-aminopropyltriethoxysilane (APES)

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