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.