Fixation is the process by which the constituents of cells and tissues are fixed in a physical and chemical state so that they will withstand subsequent treatment with various reagents with minimum loss of architecture.
Fixation is achieved by exposing the tissue to chemical compounds called Fixatives
the quality of the section in the slide is as good as the quality of the fixed tissue specimen
To preserve or maintain tissue with anatomical and structural characterization, the most important reaction is the stabilization of protein
The primary aim of fixation is to preserve the morphological and chemical integrity of the cell
The secondary aim is to harden and protect the tissue, thus easier to cut
Fixation is the first and most critical step in histopathologic techniques/steps
Fixation preserves the tissue by stopping all cellular activities so that the cells can be viewed under the microscope as if they are still in their original living state
Hypotonic Solution → cells cause to swell
Hypertonic Solution → cells cause to shrink
Additive Fixation is a mechanism where the chemical constituent of the fixative is taken in and becomes part of the tissue.
example:
Formalin → form cross-linked/molecular complexes and gives stability to the protein
Non-Additive Fixation is a mechanism where the fixing agent is not incorporated in the tissue but alters the tissue composition and stabilizes tissue.
example:
Alcohol fixatives → removes bond water that is attach on a certain grp within a molecule
Hydrogen Ion Concentration pH in fixation should be 6.8 and 8.0
Routine temperature in fixation is room temperature = 20-25°C; 0-4°C temperature for Electron Microscopy & Histochemistry
Thickness of Section in fixation for:
EM → 1-2mm
Light Microscopy → 2cm
For best result, slightly Isotonic solutions (400-450 mOsm) is used
Concentration for fixation depends on the size and number of specimen:
Formaldehyde → 10% soln
Glutaraldehyde → 3%
Immunoelectron Mic → 0.25% (ideal concentration)
Prolonged fixation may cause shrinkage and hardening of the tissue, duration should be:
2-6 hrs for small tissues
24 hrs for big tissues
3 hrs for EM
Specimen should be placed in fixative immediately to prevent autolysis and putrefaction
Standard: 4-6 hrs
Penetration of tissue should be 1 mm/hour
The recommended/standard of Tissue: 2x2 cm, not more than 4 mm thick
Volume should be 20x the tissue volume
routinely → 1:20
traditionally → 10-25x vol of tissue
Fixatives hardens soft and friable tissues, which makes handling and cutting easier; acceleration of alcohol during dehydration
Fixatives makes cells resistant to damage and distortion to achieve right osmolality
Fixatives acts as mordant or accentuator thereby facilitating staining process
Fixation reduces the risk of infection during handling and actual processing of tissue because all tissue specimen is hazardous
Simple fixatives are composed of one component substance
Simple fixative: Aldehyde
Formaldehyde
Glutaraldehyde
Simple fixative: Metallic Fixatives
Mercuric Chloride
Chromate Fixatives
Lead Fixative
Heat
Compound fixatives are made up of two or more fixatives.
Compound fixatives: According to Action
Microanatomical fixatives permits general microscopy study
Cytological fixatives preserve particular microscopic elements of the cell
Cytoplasmic fixatives never contains Glacial Acetic Acid because it will destroy Mitochondria and Golgi Apparatus; has a pH of >4.6
Nuclear fixatives always contain Glacial Acetic Acid which is a primary component due to its affinity for nuclear chromatin; has a pH <4.6
Histochemical fixative preserves chemical constituents of cells and tissues
20-22°C native DNA and RNA does not react with formaldehyde
If reaction mixture are heated: 45°C in RNA, 65°C in DNA reaction will begin.
Ethanol and methanol, including Carnoy’s solution are commonly used fixatives for nucleic acids.
Ethanol is the most usable in DNA fragment for PCR
Formaldehyde limits the size of fragments
Aldehyde fixatives are satisfactory for routine paraffin sections; for electron microscopy, Histochemical and enzyme studies
Formaldehyde or Formalin is the most common, widely used; it is a gas produced by oxidation of methanol
10% recommended, buffered to 7.0
Commercially available → 35-40% gas by weight
Pure stock solutions → >40%
Fixation time: 24 hrs at 35°C, 48 hrs at 20-25°C
10% formol saline is made up of formaldehyde diluted to 10% NaCl; recommended for CNS tissues and post-mortem tissues for histochemical exam
10% neutral buffered formalin or phosphate-buffered formalin is used for preservation and storage of surgical, postmortem and research specimens and dx pathology
Fixation Time: 4-24 hrs
Recommended and routinely used
Formol is corrosive, recommended for routine postmortem tissues
Fixation Time: 3-24 hrs
Alcohol formalin fixative coagulates mucus and can be used to fix sputum, which is why it is recommended for sputum specimens