CLEARING  produce excessive shrinkage, hardening

 Dissolve out aniline dyes

Steps in histopathologic techniques  Evaporate quickly in water bath
1. Fixation Factors Affecting Clearing
2. Dehydration
3. Clearing * Boiling point - those w low bp are more readily
4. Wax/paraffin impregnation replaced by melted paraffin
5. Embedding * Viscosity - affects the speed of penetration of the
6. Blocking clearing agent
7. Trimming * Prolonged exposure to clearing agent - causes tissue
8. Sectioning to become brittle
9. Staining
10. Mounting
11. Labeling COMMONLY USED CLEARING AGENTS

1. Xylene
Clearing (or dealcoholization) 2. Toluene
3. Benzene
- process whereby alcohol or dehydrating agent is 4. Chloroform
removed from the tissue 5. Cedarwood oil
- ---> replaced w a subs that will dissolve the wax w/c 6. Aniline oil
the tissue is to be impregnated (e.g paraffin), medium 7. Clove oil
on w/c tissue is to be mounted (e.g Canada Balsam) 8. Carbon tetrachloride

"Clearing agent" (TABLE)

- the tissue has a translucent when the dehydrating

agent has been replaced by a solvent

FUNCTIONS OF THE CLEARING AGENT

* When used after alcohol dehydration:

- Serves to mix w alcohol and remove it from the
tissue
* When used after tissue section has been stained:
- Will make microscopic tissue preparation
transparent
* When the tissue is to be cleared directly from
water:
- Will merely improve refractive index of the tissue,
(glycerin and gum syrup), no dealcoholization
involved

CHARACTERISTICS OF A GOOD CLEARING AGENT

 Be miscible w alcohol
 Be miscible w and easily removed by paraffin wax - to
facilitate penetration in the embedding process or
mounting medium - to facilitate mounting of sections
 Make tissues transparent - not all clearing agents exhibit
this property
 Infiltrating and embedding rgts - MP 46-68
- 1-2 micrometer
- paraffin wax - mixture of long chained HC Technique
- Melting pt 47-64 C * Transfer into mixture of clearing agents (3-6 hrs)
- To promote desirable ribboning during microtomy, * 3 changes of wax (3-6 hrs each)
paraffin wax of suitable hardness at room temp * 68-78 C
should be used * Heavy microtome
- Ralwax
- Histoplast
Paraffin wax additives

* It contains plasticizer or other resins

 Paraplast
* Ex-beeswax
* Rubber - Paraffin and synthetic plastic polymer's
* Ceresin - MP = 56-57 C
* Plastic polymer - More elastic and resilient
* Diethylene glycol disterate - Large dense tissue blocks s/a bones and brain
* Higher m.p than paraffin
* Inc hardness: add stearic acid * e.g. Embeddol - synthetic wax substitute similar to
* Dec mp add spermaceti or phenanthrene paraplast w a MP of 56-58 C

- It is less brittle and less compressible than paraplast

Wax additives:
* Bioloid- a semi synthetic wax recommended for
 Piccolyte 115, a thermoplastic terpene resin added at
embedding eyes
the rate of 5%-10% to the infiltrating wax, or to the final
investing paraffin wax to improve tissue support for thin * Tissue Mat is a product of paraffin, containing
sectioning and facilitate flattening ang expansion of rubber, with the same property as paraplast
sections on the water bath
 Plastic polymers s/a polyethylene wax, added to improve
adhesion, hardness and plasticity.  ALTERNATIVE EMBEDDING MEDIA

 Dimethyl sulphoxide (DMSO) reduces infiltration times and * Processing rgts remove or destroy tissue
facilitates thin sectioning, DMSO scavenges residual components that are object of investigation (e.g
lipids)
transition solvent and alters tissue permeability by
* Sections are required to be thinner (e.g lymph
substituting for or removing bound water thus improving
nodes)
infiltration * The use of heat may adversely affect tissues or
enzymes
 Paraffin wax * The infiltrating medium is not sufficiently hard to
support the tissue
* Normally used for routine work
* MP = 45 C, 52, 56, and 58 C
- Hard wax (58-60) preferred for hard tissue  RESIN
- Soft wax (42-52) for fetal and areolar tissue
- Medium wax (54-56) for routine work - Used as infiltrating and embedding medium
* Hard tissues require wax w higher MP than soft - Used for EM
tissues - Ultra thin secs for higher resolution and also
* The tissue is submerged in two or more changes of undecalcified bone
melted paraffin wax, either in a paraffin oven or in an
incubator w/c has been regulated at 55-60 C
 AGAR

* Ester wax - Above doesn't provide sufficient support for

sectioning
- Steedman (1960) - Cohesive agent for small friable pcs of tissue after fix
- Paraffin + celloidin - Agar Embedding Medium
- Harder
 - Agar-Paraffin Wax Double Embedding For * Blocks hardened by evaporation
Fragments, biopsies and friable specimen * Rubbery consistency is enough
- Agar-paraffin wax double embedding for BM * Chloroform vapours
Aspirates and cell suspension using the collodion bag * Bloch is fixed to vulcanite or hardwood
technique
- AGAR-ESTER WAX DOUBLE INFILTRATION
 Dry celloidin

 GELATIN - Overcome the disadvantage of storing in 70%

alcohol
- Gough Webtworth technique and in frozen section - Gilson's mixture (chloroform + cedarwood oil)
- Rarely used - Transparent cont and expose to air (ev)
- Frozen sections of friable or partially necrotic tissue - Air tight bottle
- Immersed in 10% formalin
- In phospholipid and enzyme studies tissues may be
infiltrated and embedding in gelatin  Double embedding
Technique
* Tissue is first impregnated in celloidin subsequently
* Tissue is fixed blocked in paraffin wax
* Wash for 6-12 hrs Peterfi's procedure
* 10% gelatin in 1% phenol (24 hrs) - Dehydrating
* 20% gelatin (12 hrs at 37 C) - Celloidin methylbenzoate mixture (2-3 days)
* Embed in 20% gelatin - Benzene 2-3 changes each 6 hrs
- Paraffin embedding

 Celloidin
 Water soluble waxed
- has a rubbery consistency allowing a continuous
shearing type of cutting w/c is beneficial for delicate - Eliminating the necessary of dehydration and
tissue clearing
- Dehydration- impregnation w LVN in sol.n - solvent - This avoiding shrinkage
w evaporate = produce block E.g
- Cox (1983) * Carbowax
Preparation * No need to be dehydrated and cleared before
infiltration
* Difficult to dissolve than LVN * Less shrinkage
* Once dissolved equal amnt of ether is added * Miles and Linder (1952)
* THIN SOLN - 5% LVN or 2% celloidin in * MP = 38-42 C or 45-46 C
ethanol/ether
* MEDIUM SOLN - 10% LVN or 4% celloidin
Technique
* THICK SOLN - 20% LVN or 8% celloidin
- Wash tissue
Processing - 50% polyethylene glycol 900 in distilled water (10-15
mins)
* 70% ethanol - 2 changes, 24 hrs each - Four changes of molten polyethylene glycol at 28-30
* 95% ethanol - 2 changes, 48 hrs each C (45 mins)
* Absolute alcohol - 2 changes over 2-5 days - Polyethylene + nonex 63B at 39 C (30-40 mins)
* THIN 3-5 days - 3 parts of nonex+ 1 part polyethylene (15 mins)
* MEDIUM 5-7 days - 3 changes of nonex at 39 degrees (30-45 mins each)
* THICK 5-7 days
Casting and blocking
Synthetic resins
* Following impregnation thick soln
* 8% celloidin - Used for ultra thin secs for EM
* Mould 1 quarter inches in depth - 0.5-2 micrometer
* L pieces can't be used - Glass knife used
* Glass petri dishes 2 inches in depth w loose fitting - Methacrylate and epoxy resins
ground glass lids - Also used for bone and teeth
  Resin embedding * Ethanol, isopropanol and proprietary mixtures of
alcohol, and paraffin
* Undecalcified bone * Xylene and formalin are not used in this process,
* 0.5-1.5 micrometer w/c eliminates toxic fumes and carcinogens
* Methyl + butyl methacrylate * Disadvantages- temps must be maintained bet 70
* Newer - 2 hydroxythyl methacrylate and glycol and 85 C, and the size of tissue sample is critical
methacrylate (2mm)
* 2 phases
* JB-4 LR white
Mtd  Ultrasound-stimulated processing

* Resin soln A monomer - The most important effect of ultrasound at

- HEMA 80 mL frequencies of 10 kHz-1 MHz is agitation.
- 2 Butoxythanol 8mL - Processing is performed in rgt containers suspended
- Bezoyl peroxide 1g in a detergent soln w/in the transducer tank of an
* Resin soln B - activator ultrasonic cleaner operated at 50 watts
- Polyethylene glycol 400 15 parts
- N-N dimethylaniline 1 part
* Fix into 10% neutral formal saline  Automated tissue processing

- The basic principle of tissue processing requires

* 70% ethanol 1 hr
exchange of fluids using a series of solns for a
* 90% ethanol 1 hr
* 100% ethanol 3 changes 30 mins predetermined length of time in a controlled
* Solution A monomer 2 hrs environment
* Fresh monomer overnight
- 12 stage cycle
* Embed
* Soln A 42 parts
* Soln B 1 part  Tissue-transfer processors
* Mix - These processors are characterized by the transfer
of tissues, contained w/in a basket, through a series
of stationary rgts arranged in-line or in a circular
 Sodium carboxy methyl cellulose (CMC) carousel plan.
- 9-10 rgt and 2-3 wax positions
- Frozen tissues are transferred from coolant directly - Of 30-110 casettes
into 5% CMC, briefly placed under vacuum to remove - Vertical oscillation or rotary motion
trapped air, then frozen to a solid block for sectioning.

 Polyvinyl alcohol (PVA)  Fluid-transfer processors

- Cross-linking PVA w glutaraldehyde provides a final
hydrophobic block containing some 10% water; w
improved sectioning characteristics and good
preservation of lipids, CHONs and carbohydrates
Microwave processing
* Precise temperature ctrl and timer, and an
interlocked fume extraction system to preclude
accidental solvent vapor ignition. Agitation is provided
by an air-nitrogen sys
- In fluid units processing fluids are pumped to and
from a retort in w/c the tissues remain stationary
- 10-12 rgt
- Temperatures adjustable bet 30-45 C, 3-4 paraffin
wax stations w variable temperature settings bet 48-
68 C, and vacuum-pressure options for each station
- 100-300 cassettes
- Tidal action

* The microwave over shortens the processing time

from hours to minutes

* Diffusion of the solns into the tissue by inc the

internal heat of the specimen