SUBSTITUTES FOR PARAFFIN WAX  ADVANTAGES:

 permits cutting of thicker sections

3. ESTER WAX  recommended for processing of neurological tissues
 melting point: 46-48°C  rubbery consistency which serves as a good support for the
 harder than paraffin tissues
 The hardness of this wax can be compared to Billiard  infiltration of dense tissues (tend to collapse due to air
ball spaces are supported better. Thereby avoiding the crumbling
of the tissues during sectioning)
 not soluble to water but soluble to 95% ethyl alcohol & other  CEDARWOOD OIL: used in dry celloidin technique to soften
clearing agents brittle area
 it can be used for impregnation without prior clearing of the  not require heating
tissue  minimum shrinkage & tissue distortion
 if with clearing agents: use Cellosolve or xylene  DISADVANTAGES:
 gradual removal of clearing agent (3-6 hours in ester wax;  very slow (days or weeks)
before 3-4 changes of pure wax)  serial sections difficult to prepare
 very flammable due to ether
 sectioning
 photomicrographs difficult to obtain
 done in heavy duty microtome due to relatively hardness of  very volatile
wax
 SLIDING or SLEDGE TYPE MICROTOME 2 methods used for celloidin impregnation of tissue:

4. WATER SOLUBLE WAXES 1. WET CELLOIDIN METHOD

 melting point: 38-42°C or 45-56°C
 plastic polymers and mostly polyethylene glycol
 added to paraffin waxes to:
 improve adhesion, hardness & plasticity
 Carbowax
 Hygroscopic (it absorb water and it dissolve during
fishing out
 polyethylene glycol solid at RT & soluble to water
 Advantages:
 not requires dehydration & clearing of tissues: reducing
processing time
 preserve neutral fats & lipids
 suitable for enzyme histochemistry and for the
preservation of cytologic details.
 FOUR CHANGES OF CARBOWAX:
 70% carbowax: 30 minutes
 90% carbowax: 45 minutes
 100% carbowax: 1 hour
 100% carbowax: 1 hour
 tissue embedded in FRESH CARBOWAX AT 50°C and
rapidly cooled in ref. temperature
 10% Polyethylene glycol 900 or addition of soap to water
 reduce tissue distortion
 promotes flattening & “floating out” of sections in water
bath
 recommended for bone, teeth, large brain sections and
whole organs
 After the usual fixation and dehydration of tissue, it is placed
in equal part of ether and alcohol for 12-24 hours.
 impregnation:
 thin celloidin (2-4%): 5-7 days
 medium celloidin (4-6%): 5-7 days, then drained off
the pour the,
 thick celloidin (8-12%): 3-5 days until the tissue
specimen has become impregnated
 embedding:
 freshly poured thick celloidin
 kept in tightly covered jar or dessicator (allows
evaporation. of alcohol-ether solvent)
 the tissue block is stored in 70-80% alcohol before cutting
 avoid dehydration & shrinkage of tissue
2. DRY CELLOIDIN METHOD
 preferred for WHOLE EYE sections
 similar to wet celloidin method except:
 70% alcohol is not used for storage
 Gilson’s mixture: made up of equal parts of
chloroform and cedarwood oil, is added to the
celloidin block before hardening to make tissue
transparent

5. DIMETHYL SULPHOXIDE (DMSO) NITROCELLULOSE

 added to proprietary blends of plastic polymer paraffin waxes  LVN (low viscosity nitrocellulose)
 Advantages:  form of celloidin soluble in equal concentrations of ether &
 reduces infiltration time alcohol
 produces thin sections  lower viscosity but has higher concentrations and still penetrates
tissues rapidly
CELLOIDIN IMPREGNATION  forms harder tissue blocks: thinner sections
 also known as COLLODION  to prevent cracks of tissue blocks: add PLASTICIZERS (oleum
 purified form of nitrocellulose ricini or castor oil)
 suitable for specimens with large hollow cavities which tends to  low viscosity nitrocellulose is more explosive than celloidin and
collapse, hard and dense tissues such as bones and teeth, large should therefore be handles with care.
tissue sections of the whole embryo
 mainly for soft tissue sections of mixed consistency (eyes & brains) GELATIN IMPREGNATION
 samples:  rarely used except when:
 2%: thin; 4%: medium; 8%: thick solutions dissolved in equal  dehydration is to be avoided
parts of ether & alcohol  Histochemical & enzyme studies and in frozen sections
 no heat required  prevents fragmentation of tough & friable tissues when frozen
sections are cut
 has rubbery block consistency (good support to tissues)  recommended for delicate tissues
 water soluble
 not require dehydration & clearing
 if we will used the gelatin, we usually add 1% phenol to prevent
the molds
 has low melting point
 not causing over-hardening of tissue due to heat
 sample preparation:
 10% formalin: wash with running water
 10% gelatin with 1% phenol: 24 hours
 20% gelatin with 1% phenol: 12 hours
 20% gelatin with 1% phenol: allowed to cool in
refrigerator temperature
 10% formalin: 12-24 hours
 tissue should not be > 2-3mm thick
 harder to freeze than non-impregnated tissues
 volume: 25x the volume of the tissue

EMBEDDING
 We will have used an embedding medium, we pour it in to the
mold and orient the slide at the center and allow it to solidify
 should match the tissue type in strength and hardness
 if too soft: sections will be torn and shredded
 if too hard: section will be brittle and shatter
 embedding: paraffin wax melted above 5-10°C
 solidify: -5°C or immersed in cold water
 ORIENTATION (it is a process in which the tissue is arranged in
precise positions in the mold)
 precise arrangement of tissue in:
 mold during embedding
 microtome before cutting
 slide before staining