Histology (Tissue processing and Staining)
Secondary Amyloidosis
(A-A) Chronic inflammatory disease states Shows - birefringence with KMnO4 pre-treating
Primarily Amyloidosis
(A-L) No presence of inflammatory disorder. Shows + birefringence with KMnO4 pre-treating
Temperature of cryostat
-20 degrees
Bouin's Fixation method
1) Formaldehyde (non-coagulating) 2) Picric acid (coagulating) 3) Acetic acid (precipitating) 12-24 hours at an acidic pH
Zinc Formalin fixation method
1) Formaldehyde (non-coagulating) 2) Zinc chloride (does not denature protein) Used up to a week at pH of 6.25 Used as a routine fixative of for immunohistochemical methods
B-5 fixation method
1) Mercuric Chloride (coagulating) 2) Sodium acetate 3) Distilled water 4) Formaldehyde (non-coagulating) Between 4-6 hours with a pH of 5.8-6.0 Best for bone marrow, renal biopsies
Zenker's Acetic fixation method
1) Mercuric chloride 2) Potassium dichromate 3) Sodium sulfate 4) Distilled water 5) Acetic acid Maximum time is 24 hours, acetic acid = acid pH Fixation of bone marrows or post-fixation
Grocott procedure
1. 5% Chromic Acid (Oxidizes carbs to aldehydes) 2. 1% Sodium bisulphite (Removes acid discoloration) 3. Working Methenamine Silver (impregnation) 4. 0.1% Gold Chloride (Toning) 5. 5% Sodium thiosulphate (Remove unreduced silver) 6. Light Green (Counterstain)
Von Kossa procedure
1. 5% Silver Nitrate (Substitutes silver for calcium) 2. 5% Sodium thiosulphate (Removes unreduced silver) 3. Nuclear Fast Red (Counterstain)
Reticulin Stain procedure
1. Acidified KMnO4 (oxidizes hexose groups) 2. 1% Oxalic Acid (removes discoloration from KMnO4) 3. 2.5% Ferric Ammonium Sulphate (sensitizes tissue) 4. Ammoniacal Silver (impregnates) 5. 10% Formaldehyde (reduces silver) 6. 0.1% Gold Chloride (tones) 7. 5% Sodium Thiosulphate (removes unreduced silver) 8. Nuclear Fast Red (counterstains)
Requirements of a counterstain
1. Be of a contrasting colour 2. Should be relatively pale (doesn't interfere) 3. Should not remove primary stain
Perl's Prussian Blue procedure
1. Bring to water 2. 2% potassium ferrocyanide and 2% HCl for 30 minutes 3. Rinse in water 4. Counterstain in Nuclear Fast Red for 5 minutes 5. Rinse in water 6. Dehydrate, clear, mount
Masson Fontana procedure
1. Bring to water 2. Silver nitrate at 56 degrees for 1 hour 3. Wash in running water 4. Tone in 0.2% gold chloride for 10 minutes 5. Wash in running water 6. 5% sodium thiosulfate for 5 minutes 7. Wash in running water 8. Counter stain in 1% Neutral red for 5 minutes 9. Wash in running water 10. Dehydrate, clear, mount
Congo Red procedure
1. Bring to water 2. Stain in Harris Hematoxylin for 2.5 minutes 3. Wash in running water 4. Alkaline salt solution for 20 minutes 5. Stain with Congo Red for 20 minutes 6. Dehydrate, clear, mount
Verhoff's procedure
1. Bring to water 2. Stain in Verhoff's until section jet black (20 minutes) 3. Rinse in running water 4. Differentiate in 2% ferric chloride (2-3 dips) 5. Rinse in water 6. Remove iodine in 5% sodium thiosulfate for 1 minute 7. Rinse in water 8. Counter stain in Van Gieson's for 3 minutes
Toluidine Blue procedure
1. Bring to water 2. Toluidine Blue stain for 5 minutes 3. Dehydrate, clear, mount
Gram Stain procedure
1. Crystal Violet for 1 min 2. Gram's Iodine (trapping agent/mordant) 3. Acetone (decolorization) 4. Basic Fuchsin (2ndard stain) 5. Gallego's Solution (Differentiation/fixation) 6. Tartrazine (Background stain)
Ziehl Neelsen procedure
1. Filtered Carbol Fuchsin (Phenol +EtOH, basic dye) 2. Acid Alcohol (Decolorizer) 3. Light Green (Counterstain)
Oil Red O procedure
1. Formaldehyde fixation with fumes 2. Dehydrate with propylene glycol 3. Stain with Oil Red O 4. Counterstain nuclei with Harris Hematoxylin 5. Mount in aqueous mountant
Luxol Fast Blue procedure
1. Luxol Fast Blue (stains Myelin) 2. 95% Alcohol (Removes excess LFB) 3. 0.5% Lithium Carbonate (Differentiates Myelin) 4. H&E staining procedure
Why isn't metallic Impregnation a true stain?
1. No Chromophore groups present 2. Silver is deposited on structures, not within
PAS/Digest procedure
1. Place one section in diastase (37 degrees) for 1 hour 2. Rinse in water 3. Place both sections in 0.5% periodic acid for 5 minutes 4. Place in Schiff's reagent for 15 minutes 5. Wash in 0.5% potassium metabisulfite 6. Rinse in water 7. Counterstain with Harris Hematoxylin for 30 seconds 8. Wash in alkaline tap water
Masson's Trichrome procedure
1. Pre-heated Bouin's fixative (Sensitizes reactive sites) 2. Weigert's Hematoxylin (Stains nuclei) 3. Acid Fuchsin (Stains cytoplasm, RBCs, collagen) 4. Phosphotungstic/molybdic acid (Differentiates stain) 5. Aninline Blue (Stains collagen) 6. 1% Acetic acid (Differentiates secondary stain)
Martius-Scarlet-Blue procedure
1. Preheated Bouin's fixation (Create reactive sites) 2. Weigert's Hematoxylin (Stain nuclei) 3. Martius Scarlet Yellow (Stains RBCs and early fibrin) 4. Ponceau 6R (Stains muscle and mature fibrin) 5. 1% Phosphotungstic acid (Differentiate out red dye) 6. Aniline Blue (Stains collagen and old fibrin) 7. 1% Acetic acid (Differentiates collagen)
What is the most common specimen fixative?
10% neutral buffered formalin
Temperature of sectioning water bath
5-10 degrees below melting point of wax
What is the pH of the diastase phosphate buffer?
6.0
Melting temperature of paraffin wax
60 degrees
Thickness of sections for Congo Red
8 micrometers
Chromogen
A coloured substance that has no affinity for fibres or tissues
Fluorochromes
A group of dyes that absorb ultraviolet or blue light and emit light of a longer wavelength. Often used to label antigen-antibody reactions
Mordants
A metallic salt added to dyes to give them affinity for tissues
Definition of Immunohistochemistry
A technique for identifying tissue constituents by means of antibody-antigen interactions (Used to diagnose and locate tumors)
Sensitivity of PAP vs. ABC in Immunohistochemistry
ABC is more sensitive than PAP because of the high affinity of avidin for biotin
Specimen pathway in lab
Accessioning --> Grossing --> Sectioning --> Staining --> Pathologist viewing
Alcian Blue results
Acid mucins, nuclei = Blue Background = Pink
Ziehl Neelsen results
Acid-fast bacilli = Red Nuclei = Blue/green Background = Green
Ziehl Neelsen methodology
Acid-fast bacilli are stained because carbol fuchsin penetrates the waxy capsule by reacting with mycolic acid
How do you test the potency of Schiff's Reagent?
Add a few drops to 5 mL of formaldehyde. An immediate bright purple-red color should be seen.
Mercuric Chloride fixation mode
Additive, coagulating. Not known to react with carbs or lipids. Not usually used alone (component of Zenker' acetic)
Glutaraldehyde fixation mode
Additive, non-coagulating Similar to Formalin Used for electron microscopy.
Formalin fixation mode
Additive, non-coagulating. Forms cross-links/bridges between the tissue. Millonig formalin may be used for electron microscopy
Why is Clearing done?
Alcohol and wax are not miscible. Serves as an intermediate step before infiltration.
Which stain for fine elastic fibers?
Aldehyde fuchsin stain
What is the purpose of Schiff's reagent?
Aldehyde groups formed oxidize the reagent
Another name for Methenamine Silver
Alkaline silver hexamine
The stain in which there is the most danger of sections floating off the slides
All silver impregnations
Degrees of metachromasia
Alpha - negative - blue (monomers) Beta - intermediate -purple (dimers/trimers) Gamma - positive - pink/red (polymers)
Heat Induced Epitope Retrieval (HIER)
Amount of antigen reactivity obtained is determined by the temperature, pH and buffers used
Role of phenol in Carbol Fuchsin dye
An accentuator
Acidic dyes
Anionic, have a (-) charge, bind with basic/acidophilic tissues (I.e. Eosin)
Two classifications of metallic Impregnation
Argentaffin and Argyrophil
Difference between an Argentaffin and Argyrophil reaction
Argentaffin can reduce silver to form visible deposits on its own. Argyrophil can impregnate but needs an external reducing agent.
Masson Fontana's other name?
Argentaffin staining
Another name for Ammoniacal silver
Argyramine
Other name for Reticulin Stain
Argyrophilic Silver Stain
= Chromogen
Benzene + Chromophore
= Autochromophoric system
Benzene + Chromophore + Auxochrome
Buffer used for Toluidine Blue
Benzoate
Which endogenous substance is used in Immunohistochemistry?
Biotin. May cause non-specific staining. Pre-treatment with avidin will saturate any endogenous biotin.
Mercuric Chloride precipitate
Black/brown, cannot be prevented. Polarizes light. Can be removed from tissues using alcoholic iodine.
Alternating thick and thin microtome sections
Blade not secure in microtome holder
Most common reason for sectioning problems, not involving the microtome itself
Block has gotten too warm while trying to section it
Significance of mast cells
Blood coagulation, allergic reactions, autoimmune disorders
Von Kossa results
Calcium deposits = Black Nuclei = Red Background = Pink
Danger of silver solutions?
Can be explosive!
Significance of elastin
Can be used in demonstrating elastic tissue atrophy, tumours in blood vessels
Bouin's fixation precipitate
Cannot be prevented due to acidic nature of fixative. 2% ammonia in 80% alcohol can remove it.
What else will the Von Kossa stain detect besides calcium?
Carbonate, phosphates, oxalates, urates, chlorides
Basic dyes
Cationic, have a (+) charge, bind with acidic/basophilic tissues (I.e. Methylene Blue)
Amphoteric Dyes
Cationic/basic below the isolelectric point and anionic/acidic above it.
Significance of Helicobacter Pylori
Causes stomach ulcers and chronic gastritis
Metachromasia
Certain chromotropic tissue components combine with dyes to produce a colour that is different from the original dye or the rest of the tissue
Mode of decalcification by EDTA
Chelating calcium out of the bone
Three main components of myelin
Cholesterol, phospholipids and cerebrosides
Groups that are added to benzene ring, can absorb light waves to produce colour
Chromophores
Another name for Reticulin
Collagen III
Acidophilic (basic) components of tissues
Collagen, RBCs,
The type of working suspension used for Oil Red O
Colloidal
Alcian Blue methodology
Copper phthalocyanine dye (basic) forms salt linkages with acid mucosubstances. At pH 2.5 both carboxyl and sulphated groups stain.
Chromogens used with Peroxidase labels
DAB = Brown AEC = Rose red/pink
How often should temperatures be checked for QC?
Daily
Significance of glycogen
Decreased in ulcerative colitis, increased in stomach cancer, tumours, and the presence of Entamoeba histolytica
Why is an aqueous mounting medium used for fats?
Dehydration and resinous mountant would dissolve the fats
Giemsa methodology
Demonstrates Helicobacter pylori using a neutral dye (combination of Azure A eosinate and Methylene Blue Eosinate)
Gram Stain methodology
Demonstrates bacteria based on the content of their cell walls. Negative bacteria have more lipid than positive bugs.
Von Kossa methodology
Demonstrates calcium using a silver substitution reaction. Exposure to light reduces silver salts to black metallic silver.
Grocott methodology
Demonstrates fungus by oxidizing carbohydrates into aldehyde groups which take up silver. Oxidation, but no reduction step.
Oil Red O methodology
Demonstrates lipids based on selective solubility. Dye prefers lipids to the solvent in which it is dissolved.
Metallic Impregnation
Deposition of salts of heavy metals, and subsequent reduction leaves an opaque, insoluble residue
Endogenous Nonhematogenous Pigments
Derived from the body but are not blood related (I.e. Melanin)
Masson's Trichrome methodology
Differentiates collagen and muscle fibers based on porosity of tissue and molecular weight of dye.
Martius-Scarlet-Blue methodology
Distinguish between early, mature and old fibrin based on porosity of the tissues to different sized dyes.
Direct staining
Dye interacts with the tissue and salt linkages are formed
Verhoff's other name
Elastic Van Giesen
Verhoff's Stain results
Elastic fibres - Black Nuclei - Black Collagen - Red Muscle and background - Yellow
Auxochromes
Enables a Chromogen to attach to tissues, and act as colour modifiers (I.e. the amino group). Ionizing radical.
Accentuators
Enhance staining or increase selectivity of a stain. May act as catalysts by changing pH. Not required.
Advantages of Mercuric Chloride fixation
Enhances staining by making tissues receptive to acid dyes. Excellent protein coagulant.
What does sensitizing tissues in an Argyrophilic silver stain do?
Enhances the contrast in the final stain
What should you do before preparing a stain?
Ensure color index, dye lot and purity match the method
B-5 fixation advantages
Excellent fixing of cellular tissue. Excellent nuclear fixation and special stain results
Advantages of Glutaraldehyde fixative
Excellent for electron microscopy due to ultra-structure preservation
Van der Waal's Forces
Exist between the electrons of one atom and the nucleus of another. Weakest of all attractive forces.
What should be done if a specimen from a patient with AIDS, TB or Hepatitis is received?
Extend fixation time to ensure fixation is complete
What is OCT media used for?
External support for cryostat specimen during cutting
Disadvantages of Mercuric Chloride fixation
Extremely toxic, causing acute nephritis. Results in layer fixation in tissue. Causes shrinkage, inhibits freezing.
The most likely cause of blue-black precipitate on an H&E
Failure to filter the hematoxylin before use
Advantages of Formalin fixative
Fast penetration rate, tolerant, inexpensive, stable. Allows less shrinkage
Oxidizing agent in Weigert's iron hematoxylin (makes it unstable)
Ferric Chloride (mordant)
Other means of demonstrating Acid-fast bacilli
Fite, fluorescent techniques
Zinc Formalin fixation Advantages
Fixation results in very fine nuclear detail. Elimination of antigens retrieval. Good for post-fixation of tissues.
Zenker's Acetic fixation disadvantages
Fixative may not be stable. Mercury precipitate will form. RBCs will be lysed, intolerant.
Why is antigen retrieval necessary in Immunohistochemistry?
Formaldehyde fixation masks antigenic sites in the tissue
Fixatives which increase basophilia
Formalin, Mercuric chloride, Osmium tetroxide
Significance of fibrin
Formed from the polymerization of fibrinogen, and is a sign of tissue damage
Formalin precipitate
Formed when formalin is acidic. Can be prevented with a phosphate buffer system.
Significance of argentaffin, melanin
Found in carcinoid tumours
Grocott results
Fungi/Pneumocystis = Black Mucins = Dark grey Background = Light green
Zenker's Acetic fixation advantages
Good nuclear fixation, rapid and even rate of penetration.
Gram Stain results
Gram positive + bugs, fungi = Purple/blue Gram negative - bugs, nuclei = Red Background = Yellow
The two stains that demonstrate fungi and mucins
Grocott's (Modified Argentaffin) and PAS
Scratches or splits in the tissue section
Hard particles/calcification in the tissue, dirty or nicked blade
Neutral dye
Has both anionic and cationic components that have an affinity for positive and negative tissues respectively.
Neutral stains
Have both Cationic and anionic portions, soluble in alcohol (I.e. Romanowsky stains)
Giemsa results
Helicobacter pylori = Dark blue Nuclei = Blue RBCs = Pink Background = Pale blue/pink
What are some natural dyes?
Hematoxylin (Heartwood of tree) Carmine (Cochineal bug) Saffron (Stigma of crocus flower)
Perl's Prussian Blue methedology
Hexaferrocyanate ion provided by the dye combines with ferric ions. The product is a ferri-ferrocyanide complex. The free ferric ions are formed when they are split from hemosiderin using HCl.
Type of silver used to demonstrate fungi
Hexamine silver in a Modified Argentaffin
Trapping agents
Hold a dye in a tissue (I.e. Iodine holds crystal violet in a gram + bug)
Enzyme labels used in Immunohistochemistry
Horseradish peroxidase and Calf Intestine Alkaline Phosphatase
Beaching agents
Hydrogen peroxide, acidified KMnO4
Lysochrome
Hydrophobic. More soluble in lipid or solvent. Not bonded to tissue, but soluble in it.
Zinc Formalin fixation Disadvantages
If Unbuffered a formalin precipitate will form. Corrosive to metal. More shrinkage of tissue than formalin alone.
Main steps in Masson Fontana
Impregnation - Ammonical silver at 56 degrees Toning - 0.2% gold chloride Removal of unreduced silver - sodium thiosulfate
Example of selective solubility
In fat staining, lysochrome dye is more soluble in lipid than in the water in its current suspension
Where are elastin fibers found?
In the walls of large arteries, in heavy sheets
Tissue sections crumble in the water bath
Incomplete clearing/infiltrating, tissue not adequately fixed before embedding
Artefact pigment
Introduced during processing or staining, appear when fixatives are used Unbuffered, or if stains are not filtered
Exogenous pigments
Introduced to the body from an outside source. Might have been inhaled, ingested or absorbed.
PAS/Digest methodology
Invokes the use of enzyme digestion to make the PAS method specific for glycogen. Diastase and alpha-amylase break down glycogen to produce smaller sugar units.
Components of Gram's iodine
Iodine, KI and distilled water
Chemical staining theories
Ionic bonds, covalent bonds, hydrogen bonding, Van der Waal's forces (ends up being a sum total of all of them)
Perl's Prussian Blue results
Iron deposits - Blue Nuclei - Red Other tissue components - shades of pink
Other Acid-fast components in tissue
Lipofuchsin granules
B-5 fixative precipitate
Mercury and Formalin precipitates. Can be removed using alcoholic iodine followed by sodium thiosulfate.
B-5 fixation disadvantages
Mercury precipitate may form. Formalin precipitate may also form. Fixative is intolerant and there are Mercury safety risks.
Zenkers's Acetic fixation precipitate
Mercury precipitate will form. No prevention, but can be removed with alcoholic iodine or decreased with 1% acetic acid.
Type of silver reaction for Grocott
Modified Argentaffin
= Lake
Mordant + Dye
Desmin antibody is a marker for...
Muscle tumors
Making up Congo red reagents
Must be made fresh and used within 15 minutes
Types of Acid-Fast bacilli
Mycobacterium tuberculosis and leprae
Luxol Fast Blue results
Myelin = Blue Nuclei = Purple Background = Pink/purple
What reagent can neutralize ammoniacal silver before disposal?
NaCl, hydrochloride
Chromogens used with Alkaline Phosphatase labels
Napthol + Fast Red = Red Nitro Blue Tetrazolium = Blue
What must be done to silver before disposal?
Neutralize with NaCl
What happens if Gram stain slides dry out?
New compounds will form in the tissue that are hard to differentiate
Oil Red O results
Nuclei - blue Neutral fats - red
Masson's Trichrome results
Nuclei = Black Collagen = Green Muscle, RBCs, cytoplasm = Red
Martius-Scarlet-Blue results
Old fibrin, nuclei = Blue Early fibrin, RBCs = Yellow Mature fibrin, muscle = Red
Order of steps in a Retic/Argyrophil stain
Oxidation, Sensitization, Impregnation, Reduction, Toning, Counterstaining
Why should Verhoff's be prepared just before use?
Oxidizers will rapidly oxidize hematoxylin to colourless hematein
What must be on histo specimen requisitions?
Patient name, hospital ID#, surgeon name, type of specimen
Bouin's Fixation Advantages
Penetrates rapidly, evenly with little shrinkage. Excellent for GI specimens, used in cytology.
Fixatives which increase acidophilia
Picric acid, Potassium dichromate
Disadvantages of Osmium Tetraoxide fixative
Poor penetration rate, expensive, blocks routine stains. Is extremely volatile and can fix the nasal mucosa.
Physical staining theories
Porosity, Adsorption, Absorption. Selective solubility
Enzyme Induced Epitope Retrieval (EIER)
Pre-treatment of sections with a proteolytic enzyme which unmasks antigen sites. Over-digestion can result in distortion.
What is 'blocking' in Immunohistochemistry?
Prevention of interference from enzymes which may be similar to the tracer and react with the chromogen, causing a false positive
Endogenous Hematogenous Pigments
Produced from heme, derived from blood
The most important factor for antigen demonstration in Immunohistochemistry
Proper fixation (10% formalin for 6-48 hours at <60 degrees)
Components of Mucin complex
Protein and carbohydrates
Two main components of amyloid
Protein with 1-2% carbs (mucopolysaccharides)
Role of Borax in Grocott's
Provide basic pH for impregnation
The most commonly found chromospheric groups
Quininiod structure, Nitro group and Azo group
Bouin's Fixation Disadvantages
RBCs are lysed, iron is dissolved. Formalin precipitate may form. Fixative is intolerant. Picric acid poses safety concerns.
Basophilic (acidic) components of tissues
RNA, DNA, cartilage matrix, mucous
Masson Fontana methedology
Reducing properties of argentaffin enables them to attract and reduce silver ions. Oxidation/reduction steps not required.
Verhoff's methedology
Regressive staining for elastic fibres. Iron hematoxylin used to over stain nuclei and elastic fibres. Tissue is differentiated with a ferric chloride mordant.
What is the purpose of potassium metabisulfite?
Remove excess reagent
What role does 1% Oxalic Acid play in the Retic stain?
Removes discoloration from KMnO4
Effect of Carnoy's fixative on Acid-fast bacilli
Removes the waxy capsule, preventing them from staining in the future
Indirect staining
Requires an intermediate substance to produce colour, such as a mordant
Two classes of mounting media
Resinous (used routinely) and Aqueous (if alcohol or xylene would hurt the stain)
Produces colour in synthetic dyes
Resonance of aromatic benzene ring
Reticulin Stain results
Reticulin fibers = Black Nuclei = Grey Background = Purple/grey/pink
Reticulin Stain methodology
Reticulin fibers are oxidized to aldehyde groups by KMnO4, allowing them to be impregnated by ammonical silver. Reduction is facilitated by formalin.
How to correct over-differentiation in Verhoff's?
Rinse in 70% alcohol and re-stain
Holes in tissue sections, moth-eaten appearance
Rough trimming too much of the block off at once, pulled out bits of tissue
What do the A and S on histo specimens mean?
S = Surgical, A = Autopsy
Dyes that need refridgeration
Schiff's, Aldehyde fuchsin, Methyl green, methenamine silver nitrate
The focus of post-mordanting
Secondary fixation
PAS/Digest results
Sections not treated with diastase = Glycogen is magenta (+) Sections treated with diastase = No staining (-)
Mounting media RI (Refractive Index)
Should be as near as possible to glass (1.518)
Disadvantages of Glultaraldehyde fixative
Shows marked shrinkage and hardening of tissues. Can only used on thin pieces.
Methods of differentiation
Simple solvents, acid differentiation, mordant differentiation, action of other dyes, action of bases, action of oxidizers
Disadvantages of Formalin fixative
Slow rate of fixation, may cause dermatitis, can produce precipitate. Picric acid may explode.
How should tissues be embedded?
So the hardest part hits the blade last. On end or on edge as appropriate.
Cleanup protocol A
Soak dissection instruments in lab detergent with phenolic disinfectant
Cleanup protocol B
Soak dissection instruments in phenolic disinfectant followed by hot, soapy water wash
Vital staining
Staining of living tissues through the phagocytosis of dye (I.e. Reticulocyte staining)
Congo Red results
Stains amyloid pink/red, nuclei blue Polarized light makes birefringent amyloid look apple green
Toluidine Blue methedology
Stains carbohydrate polymers such as heparin which are found in Mast cells. Uses metachromatic, basic dyes.
Masson Fontana results
Stains melanin black, argentaffin granules black, nuclei and other structures red
Luxol Fast Blue methodology
Stains normal myelin and Nissel substance using a copper phthalocyanine dye in the presence of lipoproteins.
Toluidine Blue results
Stains nuclei blue, mast cells purple, mucins and collagen reddish-purple. RBCs do not stain
What do you do if you over-impregnate an Argyrophil stain?
Start over with new tissue
Sections cling to microtome, block or hand
Static electricity, low humidity in the cutting room
Factors that influence staining procedures
Temperature, type of fixation, pH of water, thickness of section
Clearance angle
The angle formed by the block face and the cutting facet of the knife
Acidic or basic when referring to a dye means...
The colored portion of the dye molecule
Metallic substitution
The demonstration of calcium salts. Light is used as a reduced to blacken the silver.
Direct Immunohistochemistry staining
The primary antibody is conjugated directly to the labeling molecule
Regressive staining
The tissue is first overstrained, and then the excess stain is removed
Giemsa stain dye type
Thiazine dye
Microtome section is wrinkled, jammed or compressed
Tissue block too warm, knife blade too dull, clearance angle too small
Argentaffin
Tissue possess the capacity to reduce silver salts without the aid of a reducing agent. A modified version can demonstrate fungus.
Argyrophil
Tissues have affinity for silver salts, but visible deposits cannot be seen unless a reducing agent is added
Progressive staining
Tissues stained in sequence, the appropriate colour is obtained at the end of the sequence
Why are tissues infiltrated with wax?
To provide external support for tissues during cutting
Fatty acids are used by the liver to synthesize...?
Triglycerides, phospholipids and lipoprotein
ABC Immunohistochemistry procedure
Unconjugated primary antibody is applied to tissue to bind with antigens. Then biotin-conjugated anti-mouse IgG is added as the second layer.
How long can a stain be kept?
Unless otherwise directed, until they lose their staining properties
What can be done to intensify protein staining?
Use an acidic solution
Osmium Tetraoxide fixation mode
Used in light and electron microscopy for lipid fixation. Able to fix unsaturated lipids using oxidation. Often follows Glutaraldehyde fixation.
Significance of hemosiderin
Used to demonstrate iron deposits in hemochromatosis (abnormal accumulation). Increased iron stores are found in liver storage disease and cirrhosis. Hemorrhage and infarcts can also affect.
Congo Red methedology
Uses alkali salt solution to release H+ ions from amyloid, allowing acidic dye to bind
Which stain for coarse elastic fibers?
Verhoff's Van Gieson stain
Effect of acid fixatives on calcium salts
Washes them out of the tissue
Tissue sections attach to block on the upstroke of the blade
Wax built up on the back of the blade, clearance angle too small
Glutaraldehyde precipitation
Will form if solution becomes acidic.Unbuffered version is not used so precipitate not an issue.
An advantage of Periodic Acid
Will not easily over-oxidize
Zinc Formalin fixation precipitate
Will precipitate during dehydration. Low pH (acidic) may encourage precipitation. Can be prevented either by buffering or with acid water rinses beforehand.