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TISSUE PROCESSING
I
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Additive fixation
 The chemical constituent of the fixative is taken in and becomes part of the tissue by
forming cross-links or molecular complexes and giving stability to the protein
 Formalin
 Mercury
 Osmium tetroxide
2
...
Alcoholic fixatives

Main factors involved in fixation
1
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temperature
 Room temperature
 40C
 0-4C-EM
 60C for rapid fixation
 100C for tuberculosis
3
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osmolality
 Slightly hypertonic solutions gives the best results
 400-450mOsm
5
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25% glutaraldehyde for immuno-electron microscopy
6
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speed
 Place specimen in fixative ASAP
2
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volume
 10-25 x that of the tissue volume
4
...
They harden soft and friable tissues and make the handling and cutting of sections easier
...
They make cells resistant to damage and distortion caused by hypotonic and hypertonic
solutions used during tissue processing
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They inhibit bacterial decomposition
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They increase optical differentiation of cells and tissue components
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They act as mordants or accentuators topromote and hasten staining or they may inhibit
certain dyes in favor of another
...
They reduce risk of infection during handling and processing
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Simple fixatives
1
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formaldehyde
 B
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metallic fixatives
 A
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chromate fixatives
 Potassium dichromate
 Chromic acid
B
...
Microanatomical fixatives
Permit general microscopic study of tissue structures
 1
...
10% neutral buffered formalin
 3
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formol sublimate(formol corrosive)
 5
...
Zenker-formol(Kelly’s solution)
 7
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Brasil’s solution
B
...
Nuclear Fixatives
 Usually contain glacial acetic acid as primary component due to affinity to nuclear
chromatin
 pH of 4
...
Cytoplasmic Fixatives
 Flemming’s fluid without acetic acid
 Kelly’s fluid
 Formalin with “post-chroming”
 Regaud’s fluid(Muller’s fluid)
 Orth’s fluid
3
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formol saline 10%
 B
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acetone
 D
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Karnovsky’s solution
 Paraformaldehyde-glutaraldehyde mixture
2
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ALDEHYDE FIXATIVES
 A
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10% Formol-Saline
 C
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Formal-Corrosive (Formal-Sublimate)
 E
...
Glutaraldehyde
II
...
mercuric chloride
 The most common metallic fixative
 A1
...
Zenker-formol(Helly’s solution)
 A3
...
skin
 A4
...
chromate fixatives
 B1
...
potassium dichromate
 B3
...
Orth’s fluid
C
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PICRIC ACID FIXATIVES
 A
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Brasil’s Alcoholic Picroformol
IV
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ALCOHOL FIXATIVES
 A
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Isopropyl alcohol 95%
 C
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Carnoy’s fluid
 E
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OSMIUM TETROXIDE(OSMIC ACID)
A
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Flemming’s solution without acetic acid
VII
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ACETONE
IX
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5-3% potassium dichromate for 24 hours to act as mordant for
better staining effects
Washing-out
 The process of removing excess fixative from the tissue after fixation in order to
improve staining
 1
...
50-70% alcohol
 3
...
RETARDED BY:






1
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presence of mucus
3
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presence of blood
5
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ENHANCED BY:
 1
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agitation

FIXATION ARTIFACTS
 Formalin pigment
 “crush artifact”

Microwave technique
 Similar in mechanism to vacuum,oven (heat) and agitation to increase the
movement of molecules and accelerate fixation

 Also accelerate staining,decalcification, immunohistochemistry and
electron microscopy
Immunofluorescence And Immunoperoxidase Techniques
 For the demonstration of various antibodies
 Enzyme histochemistry
 Electron microscopy

Enzyme histochemistry
 General aim is to preserve the maximum enzyme activity at its original localization,
while also preserving integrity
 Fixed in 4% formaldehyde or formol saline
 Frozen sections may be fixed in acetone or formaldehyde and washed prior to staining

Electron microscopy





Osmium tetroxide
Glutaraldehyde
Paraformaldehyde
4C



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