Fixation Is the fundamental step in tissue processing. It has to be complete and adequate The principle of fixation: The principle involved is:• the denaturation or• precipitation (coagulation) of the protein in the tissue The process converts the tissue protein from colloidal (semi sol) in to a solid (semi gel) state Precipitation involves the appearance of mixture of granular or reticular solids suspended in a fluid. Coagulative fixative ( chromic, acetone) transforms the protoplasm of the cell into a network of reticulum which will allow: The embedding wax to pass throw the tissue Good quality of sectioning

The process converts the tissue into inert spongy mass, which makes it more rapidly permeable to stains Fixation takes place from the outer surface of the specimen towards its center Cell membrane is killed and looses its property of semi permeability, it will be no longer able to regulate osmotic gradient within and outside the cell Fixative should provide more stable bonds, which will hold tissue molecules together during dehydration and subsequent treatment

Protect the tissue from disruption during embedding and sectioning The aim of fixation: The aim of fixation is the preservation of shape, structure, relationship and chemical constituents of the tissue (physically and chemically) in as life-like manner as possible. To preserve the relationship between tissues components with minimum alteration from the living state Protect the tissue from disruption during embedding and sectioning Prepare the tissue for subsequent treatment (staining) Fixation is of selective rather than of general effectiveness, to study lipid, one would select osmium tetroxide as a fixative and avoid potassium permanganate

The criteria an ideal fixative: Works quickly with minimum distortion, penetrate the tissue rapidly, and evenly It should have no hypotonic or hypertonic effect on tissue (causes minimum shrinkage or swelling) It should coagulates the tissue proteins (e.g. protoplasmic material) to prevents the diffusion of tissue substances Inhibit putrefaction and autolysis of tissue Induce tissue hardening to facilitate cutting Protect the tissue from harmful effect of subsequent steps (e.g. dehydration) Mordanting (enhance staining method) Preserve the color (no color change), especially in the museum It should Not produce artifacts not harmful to the user

The criteria an ideal fixative….. 12. Not harmful to containers 13. It should not need treatment after fixation 14. Easy to make and not expensive

Types of fixatives: 1- Simple fixatives 2-Compound fixatives contain one chemical substance which has its own particular effect or action on one or more of the tissue substances or constituents Consists of two or more of the simple fixatives

Non-protein precipitate (denaturing) 1- Simple fixatives Protein precipitate Non-protein precipitate (denaturing) Mercuric chloride Chromic acid Acetic acid Ethyl alcohol K cl (less than 3.7 pH) Formaldehyde Osmium tetroxide K cl (less than 3.7 pH)

2-Compound fixatives Microanatomical: B) Cytological: 10% formal-saline Zenker’s solution Bouin’s solution heidenhain’s solution B) Cytological: Nuclear Flemmings' fluid Carnoy’s fluid ii) Cytoplasmic Flemmings' fluid W/O acetic acid Hellys fluid Formalin fixation with post-chroming

Hints: Bouin and 10% formalin are only microanatomical fixative Carnoy is only nuclear fixative Zenker is microanatomical and nuclear fixative Helly is microanatomical and cytological (nuclear and cytoplasmic) 10% buffered formalin and formal sublimate are the same Histochemical fixataves is different from all these types

Histochemical fixatives: Electron microscope fixatives: Used for histochemical investigations (e,g. for enzymes 1o% buffered formalin Cold acetone Cold absolute alcohol Electron microscope fixatives: Glutaraldehyde Acetaldehydes acrolein Osmic tetroxide Vapour fixatives: Used when wants to avoid liquid fixatives Fixation is possible by heating some liquid fixatives to get their vapour Formaldehyde vapour-50-80 Acetaldehyde vapour about 80 Glutaraldehyde vapour (50% aqueous)-60-80