Fixation Table.pdf

Reference: Bruce-Gregorios, J., & Faldas, M.-E. (2017). Histopathologic Techniques (US) Table 1 ALDEHYDE FIXATIVES Fixative Description...

Reference: Bruce-Gregorios, J., & Faldas, M.-E. (2017). Histopathologic Techniques (US) Table 1 ALDEHYDE FIXATIVES Fixative Description Other Information Formaldehyde (Formalin) Pure stock: 40% Working solution: 10% Neutral Buffered Formalin (10%) Best general tissue fixative (prevent autolysis and preserves tissue and cellular Advantages: cheap, readily available, compatible with many stains, morphology) penetrates tissue well, frozen sections can be prepared with ease Disadvantage: cause irritation of mucosa and if unbuffered: may reduce basophilic and eosinophilic staining of cells and may form brown pigment granules on blood containing tissue 10% Formal-Saline 40% formaldehyde diluted with 10% sodium chloride Advantages: preserve microanatomic and cytologic details with Recommended for CNS tissues, general post-mortem tissues for histochemical minimum shrinkage and distortion, preserves enzymes and examinations, lipids (phospholipids) nucleoproteins, demonstrates fats and mucin Ideal for silver impregnation staining technique Disadvantage: metachromatic reaction of amyloid is reduced Glutaraldehyde (2%; Cold and buffered) Larger molecule than formaldehyde (slower rate of diffusion) Specimen thickness should not be more than 1mm Fixes well at 4 deg. C.; gives best overall cytoplasmic and nuclear detail Advantages: more stable effect on tissues with firmer texture (CNS); Not used in immunohistochemical staining as it alters protein structures preserves plasma proteins, and suitable for fixation of cellular Has 2 Reactive aldehyde groups separated by 3 carbon atoms structures Disadvantage: expensive, penetrates tissue slowly, makes renal biopsy tissues brittle, reduces PAS positivity of reactive mucin Karnovsky’s Fixative 4% Paraformaldehyde, 1% glutaraldehyde in 0.1 M Phosphate buffer; must be prepared fresh For electron microscopy and light microscopy (samples are embedded in resin) ALCOHOLIC FIXATIVES Denatures proteins and not used routinely (causes brittleness and hardness) Good for cytologic smears- act quickly and give nuclear detail PAP smears- spray cans of alcohol fixatives Methyl alcohol (100%) Used for bone marrow and blood smear Advantages: fixes and dehydrates at the same time Isopropyl alcohol (95%) Used for touch preparations Ethyl alcohol (70-100%) Used as a simple fixative Disadvantages: Strong reducing agent (not to be mixed with chromic Used in histochemistry (nucleoproteins & nucleic acids) acid, potassium dichromate, & osmium tetroxide); lower Time: 18-24 hours concentrations cause RBCs to lyse; causes polarization (glycogen Preserves but does not fix glycogen granules tends to move towards the poles or end of cells) 1 |L.J. Gumahad 2 0 2 4 Reference: Bruce-Gregorios, J., & Faldas, M.-E. (2017). Histopathologic Techniques (US) Carnoy’s Fixative Most rapid fixative; Time: 1-3 hours (used for urgent biopsy specimens for paraffin Advantages: suitable for small tissue fragments processing w/in 5 hours) Used to fix chromosomes Disadvantage: causes RBC hemolysis, cause excessive hardening Preserves Nissl granules and cytoplasmic granules and shrinkage, slow penetrating for large tissues, dissolves acid- Excellent fixative for glycogen soluble cell granules and pigments Used to fix brain tissue for the diagnosis of rabies Clarke’s solution Used in frozen sections and smears Time: 3-4 hours Alcoholic Formalin Can be used in fixing large fatty specimens (e.g. breast) Gendre’s Fixative 95% ethanol with picric acid and glacial acetic acid Disadvantages: causes partial lysis of RBC Good for preservation of glycogen and for micro-incineration techniques Used to fix sputum (coagulates specimen) Newcomer’s Fluid Used in fixing mucopolysaccharides and nuclear proteins Time: 12-18 hours at 3 deg. C METALLIC FIXATIVES Unknown mechanism of action Increases staining brightness and gives excellent nuclear detail Best for fixing hematopoietic and reticuloendothelial tissues Mercuric Chloride Most common (saturated aqueous solution of 5-7%) Disadvantages: rapidly hardens outer layer of tissue rendering Used as a secondary fixative incomplete penetration and fixation Usually penetrates slowly and produces shrinkage of tissues Tissue fixed may contain black precipitate (removed before staining; sections are treated with 0.5% iodine solution in 70% ethanol for 5-10 minutes, decolorized for 5 minutes in 5% sodium thiosulfate and washed in running water) Routine fixative for preservation of cell detail in tissue photography Permits brilliant metachromatic staining Zenker’s Solution Recommended for trichome staining Disadvantages: does not permit cutting of frozen sections; causes Recommended for congested specimens (e.g. lung, heart, blood vessels) and gives mercuric deposits but can be removed by de-zenkerization (water-> good results with PTAH and trichome staining Lugol’s iodine -> water -> sodium thiosulfate -> water) Zenker-Formol (Helly’s) Solution Used for fixing bone marrow, extramedullary hematopoiesis, intercalated discs of cardiac muscles, and blood containing organs Lillie’s B-5 Fixative 4% aqueous formaldehyde with 0.22M mercuric chloride (ensures rapid structural stabilization) and 0.22M acetic acid (coagulation of nuclear chromatin) For bone marrow biopsies Time: 4-8 hours 2 |L.J. Gumahad 2 0 2 4 Reference: Bruce-Gregorios, J., & Faldas, M.-E. (2017). Histopathologic Techniques (US) Heidenhain’s Susa Solution For fixing tumor skin biopsies Excellent cytologic fixative OXIDIZING AGENTS Includes permanganate fixatives, potassium dichromate, chromic acid, and osmium tetroxide Used as a secondary fixative Forms crosslinks that stabilize tissue structure but causes extensive denaturation Osmium tetroxide (Osmic acid; OsO4) Powder which dissolves in water Disadvantage: expensive, can cause irritation, inhibits hematoxylin Traditionally used in EM as a fixative and heavy metal stain and counterstaining poor Fixes conjugated fats and lipids permanently (makes it insoluble during subsequent treatment with alcohol and xylene) Preserves cytoplasmic structures (e.g. Golgi bodies & mitochondria) Fixes neurological tissues (e.g. myelin and peripheral nerves) Flemming’s solution Has glacial acetic acid Most common chrome-osmium acetic acid fixative Excellent fixative for nuclear structures and permanently fixes fat Time: 24-48 hours Flemming’s solution without acetic acid Recommended for cytoplasmic structures (mitochondria) Time: 24-48 hours CHROMATE FIXATIVES Chromic acid (1-2% aq. Solution) Preserves carbohydrates Potassium dichromate (3% aq. Solution) Preserves mitochondria Regaud’s (Muller’s) Fluid Demonstrates chromatin, mitochondria, mitotic figures, Golgi bodies, RBC, and colloid- containing tissues Time: 12-48 hours Orth’s Fluid Recommended for study of early degenerative processes and tissue necrosis Demonstrates Rickettsia and other bacteria PICRIC ACID FIXATIVES Penetrates tissue well to react with histones and basic proteins Good fixative for connective tissue Bouin’s solution Recommended for fixation of embryos and pituitary biopsies Causes RBC hemolysis and reduces amount of demonstrable ferric Excellent fixative for glycogen demonstration iron in tissue Gives good results with tissue that subsequently stained with trichrome (Suitable for Mallory’s, Heidenhain’s, or Masson’s staining method) Time: 4-18 hours Hollande’s Solution Recommended for gastro-intestinal tract specimens and endocrine tissues 3 |L.J. Gumahad 2 0 2 4 Reference: Bruce-Gregorios, J., & Faldas, M.-E. (2017). Histopathologic Techniques (US) Has decalcifying properties Time: 4-18 hours Gendre’s solution Alcoholic Bouin’s solution that improves upon ageing Preferred fixative for tissues stained by Masson’s trichrome for collagen, elastic or connective tissue Brasil’s Alcoholic Picroformol Fixative Excellent fixative for glycogen OTHER FIXATIVES Glacial Acetic Acid Precipitate DNA- preservation of nuclei (chromosomes, chromatin materials) Michel’s Solution Not a fixative Not suitable for transporting cells for flow cytometry or for fluorescent Transport medium for fresh unfixed tissues that will undergo frozen section and in-situ hybridization immunofluorescence studies 4 |L.J. Gumahad 2 0 2 4