Fixation Flashcards
Acetic acid
Concentration: About 5% Fixation time: Overnight Additive: No Coagulant: No Hardens: No Acid dyes: Improves Basic dyes: Reduced Aftertreatment: None Carbohydrates: no effect Lipids: no effect Preserves nuclei Tissues shrink after dehydration if used alone
Acetone
Concentration: Absolute Fixation time: A few hours Additive: No Coagulant: Yes Hardens: Yes Acid dyes: Neutral Basic dyes: Neutral Aftertreatment: None Carbohydrates: not fixed Lipids: not preserved Poor preservation, best for enzymes
Chromic acid
Concentration: 1 - 3% Fixation time: Several hours Additive: Yes Coagulant: Yes Hardens: No Acid dyes: Improves Basic dyes: Reduced Aftertreatment: Wash well Carbohydrates: oxidized Lipids: oxidized Strong oxidizer, combines with reducing agent
Potassium dichromate
Concentration: 1 - 3% Fixation time: Hours to weeks Additive: Yes Coagulant: No Hardens: Moderately Acid dyes: Improves Basic dyes: Reduced Aftertreatment: Wash well, can cause dark precipitate when transferred to ethanol Carbohydrates: no effect Best for lipids If ph is above 3.8
Ethanol
Concentration: Absolute Fixation time: Several hours Additive: No Coagulant: Yes Hardens: Yes Acid dyes: Neutral Basic dyes: Neutral Aftertreatment: None Carbohydrates: not fixed unless attached to proteins Glycogen well preserved Lipids: not preserved, may dissolve Poor cytoplasmic preservation Fixes proteins through dehydration and precipitation
Formaldehyde
Concentration: 4% aqueous Fixation time: Days Additive: Yes Coagulant: No Hardens: Yes Acid dyes: Not enhanced Basic dyes: Not enhanced Aftertreatment: None Carbohydrates: only fixes protein part Lipids: most dissolve through clearing Causes proteins to cross link preserving morphology Cleared with ethanol to removes salts after fixing Reacts with nitrogen in the proteins
10% Neutral Buffered Formalin
10% formalin buffered with sodium dihydrogen phosphate and disodium hydrogen phosphate to pH 7.0
Reduces formalin pigment formation with bloody tissues
Glutaraldehyde
Concentration: 4% aqueous Fixation time: Several hours Additive: Yes Coagulant: No Hardens: Yes Acid dyes: Not enhanced Basic dyes: Not enhanced Aftertreatment: aldehyde block needs to be used when staining for PAS Carbohydrates: not fixed, proteins attached will be Lipids: not fixed Used for electron microscopy Adds free aldehyde groups to the fixed tissue; can pose problems for immunolabeling
Mercuric chloride
Concentration: Varies, often 6% Fixation time: 30 minutes to hours Additive: Yes Coagulant: Yes Hardens: Yes Acid dyes: Improves Basic dyes: Improves Aftertreatment: Rinse with Iodine to remove mercury pigment Excellent preservation
Methanol
Concentration: Absolute Fixation time: Several hours Additive: No Coagulant: Yes Hardens: Yes Acid dyes: Neutral Basic dyes: Neutral Aftertreatment: None Carbohydrates: not fixed Lipids: not rpeserved, may dissolve Fixes proteins through dehydration and precipitation
Osmium tetroxide
Concentration: Up to 1% Fixation time: A few hours Additive: Yes Coagulant: No Hardens: No Brittle: No Acid dyes: Poor Basic dyes: Good Aftertreatment: Wash well Carbohydrates: unaffected Lipids: preserved, reacts with double bond of unsaturated lipids, rendering them resistant to extraction by many solvents Excellent preservation Acts as mordant for lead staining, hematoxylin Penetrates poorly, need small tissue Reacts with C=C in unsaturated fatty acid chains of phospholipids
Trichloroacetic acid
Concentration: About 2% Fixation time: A few hours Additive: Yes Coagulant: Yes Hardens: Yes Aftertreatment: None Carbohydrates: not affected Lipids: not affected Fixes by reactions between negatively charged chloroacetate anion and positively charged amino groups of proteins
Zinc chloride
Concentration: Varies, perhaps 2-5% Fixation time: one or more hours Additive: Yes Coagulant: Yes Hardens: Yes Acid dyes: Some improvement Basic dyes: Some improvement Aftertreatment: None Alternative to mercuric chloride but inferior
B5
Ingredients: Mercuric chloride DI Water Sodium Acetate 40% Formalin Clear with iodine Improves nuclear and cytoplasmic staining Used with trichromes
Bouin’s picro formal acetic
Ingredients: Formalin Picric acid Clear with 70% ethanol Used as secondary fixation after formalin Used with trichromes
Duboscp-Brasil
Ingredients: Picric acid Ethanol Formalin Acetic acid Increases staining of acid dyes Recommended for glycogen
Carnoy’s
Ingredients: Ethanol Acetic acid Chloroform Clear with absolute ethanol Very rapid fixation Preserves glycogen
Clarke’s
Ingredients: Ethanol Acetic acid Clear with absolute ethanol Fast fixation, suitable for smears Used for cryostat sections
Alcoholic formalin
Ingredients: Formalin Ethanol Tap water Calcium acetate, used to reduce formalin pigment Prevents tissue from freezing
Gendre’s
Ingredients: Picric acid Formalin Acetic acid Recommended for glycogen Alcoholic Bouin's
Helley’s
Ingredients: Mercuric chloride Potassium dichromate Sodium sulphate DI water Formalin Wash well after treatment Routine fixative, excellent cytoplasmic fixation
Hollande’s
Ingredients: Water Cupric acetate Picric acid Formalin Acetic acid Not used very often Can act as mordant for some dyes including hematoxylin, good preservation
Muller’s
Ingredients: Potassium dichromate Potassium sulphate DI water Wash well after treatment May be used as secondary fixative
Formalin ammonium bromide (FAB)
Ingredients: Formalin Tap Water Ammonium bromide Used for central nervous tissue Great for silver and gold impregnations
Marchi
Ingredients: Potassium chlorate Potassium sulphate DI water Osmium tetroxide Used to demonstrate degenerating myelin
Orth’s
Ingredients: Potassium dichromate Potassium sulphate DI water Formalin Wash well with water after treatment Fixation carried out in the dark
Susa
Ingredients: DI water Mercuric chloride Trichloracetic acid Sodium chloride Formalin Acetic acid Rinse with iodine Cannot be used with Weigert's elastic fibre Secondary fixation after 10% formalin
Zenker’s
Ingredients: Mercuric chloride Potassium dichromate Sodium sulphate DI water Acetic acid Rinse with iodine Excellent morphological preservation
Zinc formalin
Ingredients: Formalin Zinc chloride Sodium chloride DI water Wash well with water
Zamboni’s
Ingredients: Paraformaldehyde Picric acid Sodium phosphate DI water Used for electron microscopy
Additive
Agents that combine with proteins
Form cross-links with their targets
Can be used with EM or LM
Coagulant
Agents that precipitate proteins
Do not fix carbohydrates or lipids
Only used for LM
Putrefaction
Bacterial attack, can be prevented with aseptic techniques or fixation
Autolysis
Enzyme attack, can be prevented with fixation
Factors influencing fixation
Temp, size, volume, time
Volume Ratio
Fixative volume should be 15 to 20 times greater than tissue volume
Aqueous fixatives/ingredients
Acetic acid Formaldehyde Glutaraldehyde Mercuric chloride Osmium tertoxide Picric acid Potassium dichromate Zinc salts
Nonaqueous fixatives/ingredients
Acetone
Alcohol
Formalin Fixation Pigments
Brown, granular, doubly retractile deposit
To remove, treat the deparaffinized and hydrated sections with:
Absolute alcohol saturated with picric acid
or
70% alcohol with ammonium hydroxide
Mercury Pigment
Dark brown to black crystals in the tissue
Treat tissue with iodine during dehydration
or
Treat sections with iodine-thiosulphate sequence
Acids
Precipitate proteins
Do not fix carbohydrates or lipids
LM only
Methylene bridge
Formed between two reactive atoms in proteins that are very close together
-CH2-
Mercaptides
Result of adding organomercuric salts to neutral formalin used for fixation was found to protect protein thiols from autoxidation,
Picric acid
Concentration: Often saturated Fixation time: Several hours Additive: Yes Coagulant: Yes Hardens: No Brittle: No Acid dyes: Improves Basic dyes: Reduced Aftertreatment: 70% ethanol Carbohydrates: no reaction Lipids: not affected Best for trichrome stains Forms picrates with AA and causes proteins to precipitate Tissue shrinks, not used for kidneys
