STAINING

Staining

Process of applying dyes on the section to see and study the architectural pattern of the tissue and physical characteristics of cells.

Histological Staining

• Involves demonstration of cytoplasm and nucleus of cells.

• Tissue components are demonstrated by direct interaction with a dye or staining solution.

• Active tissue component is colored.

Histochemistry Staining

Demonstrate the staining of chemical components.

i.e. fats carbohydrates

Types of Histological Staining

• Micro-Anatomical Stains

• Bacterial Stains

• Specific Tissue Stains

Example of Histological stains

• Wright’s

• Giemsa stain

• Methylene Blue

• Carbol fuchsin

• Crystal Violet

Immunohistochemistry

• Do not demonstrate the cells.

• Do not color the tissues.

• Detection of phenotypic markers that are detected by antibodies.

• Demonstrate tissue antigens; combination of immunologic and histochemical staining.

• Monoclonal Antibody

• Polyclonal Antibody

• Fluorochrome dye

• Colloidal Gold

• Radioisotopes

Immunohistochemistry used what type of labels?

TRUE

TRUE or FALSE

Immunochemistry used Enzyme to label to identify the antigen to tissue.

TRUE

TRUE or FALSE

Immunofluorescence used Flourochrome to label to identify the antigen to tissue.

Example of Histochemical stains

• Peri’s Prussian blue reaction

• Periodic Acid Schiff

• Sudan Dyes

• Sudan Black

• Sudan III

Regressive staining method

Hematoxylin & Eosin Staining used what type of staining?

Direct Staining

A staining technique that uses aqueous or alcoholic dye solutions to directly color tissues.

Used 1 dye.

FALSE

TRUE or FALSE

Progressive staining would cause tissue to be overstained.

Regressive Staining

A staining method where tissues are overstained, requiring decolorization to remove excess dye.

Acid Alcohol

Commonly used decolorizing agent.

Metachromatic Staining

• Involves use of a dye that when applied to the tissue will produce a different color, different color from the dye itself.

• Staining tissue with a color that is different from the stain.

• To better demonstrate epithelial mucins, amyloid, cartilage, and mast cell granules

Orthochromatic Staining

When the dye is applied to the tissue, it produce a color not exactly the same as the color of the dye but of the same shade, same hue as that of the dye.

Counterstaining

Involves the use of another dye; there will be contrast and background.

Involves application of a different color to differentiate

Metallic Impregnation

• Tissue elements are demonstrated NOT by stains but by colorless solutions of metallic salts.

• Metallic salts can leave black deposits on the surface.

Vital Staining

Dealing with fresh viable cells

Intravital stain

Injecting the dye to any part of the living body.

Supravital stain

Applied immediately to tissue after its removal from living body.

Staining Containers

• Staining Jar

• Couplin Jars

• Slotted staining dishes

Deparaffinization

Initial xylene bath is for?

Descending order of concentration

Hydrating the tissue used what order of alcohol concentration?

Ascending order of concentration

dehydrating the tissue used what order of alcohol concentration?

Hematoxylin

What is the primary dye of the H&E stain.

A nuclear stain classified as basic dye.

Acts as decolorizer of H&E staining.

Acid Alcohol role in H&E staining?

Ammonia Water

Serves as the bluing agent.

Eosin

The counterstain used for cytoplasmic staining which is an acid dye.

Blue to Black

Identify the color result of H&E staining

Nuclei

Dark Blue

Identify the color result of H&E staining

Karyosome

Pale Pink

Identify the color result of H&E staining

Cytoplasm

bright orange red

Identify the color result of H&E staining

RBCs, eosinophilic granules, keratin

Purplish Blue

Identify the color result of H&E staining

Calcium and decalcified bone

Pink

Identify the color result of H&E staining

Decalcified bone matrix collagen and osteoid

Deep Pink

Identify the color result of H&E staining

Muscle fibers

Staining Methods for Frozen Section

• H&E

• Alcoholic pinacyanol method

Natural Dyes

Derived from plants and animals like:

• Hematoxylin

• Cochineal dyes

• Orecein

• Saffron

Hematein

The active coloring agent of hematoxylin.

Hematoxylin

• Not a stain but an active coloring agent.

• It is formed from the oxidation of hematoxylin either by exposure to sunlight or ripening (slow process) or by adding oxidizing agents (faster ripening).

Alum Hematoxylin

Uses potassium alum as mordant.

Erlich’s Hematoxylin

Uses Sodium iodate

Harris Hematoxylin

Uses Mercuric oxide used in exfoliative cytology can also be used to demonstrate sex chromosomes.

Cole’s Hematoxylin

Uses alcoholic iodine as ripening agent.

Mayer’s Hematoxylin

Uses Sodium iodate With citric acid & chloral hydrate as preservative Form of hematoxylin preferred for Immunohistochemistry.

Delafield’s Hematoxylin

Uses Glycerol to stabilize oxidation process It smells like wine and gives a purple to red shade/ hue.

Gill’s Hematoxylin

Uses Sodium iodate Used for staining mucin in goblet cells.

Carazzi’s Hematoxylin

Uses Potassium iodate.

Iron Hematoxylin

Used in general for photomicrography.

Weigert’s Hematoxylin

• For muscle fibers and CT

• composed of ferric ammonium chloride mordant and ripening agent.

Heidenhain’s Hematoxylin

• For nuclei & cytoplasmic inclusion

• Composed of Ferric ammonium sulfate Mordant and Ripening agent.

Copper Hematoxylin

demonstrate spermatogenesis

Tungsten Hematoxylin

form of hematoxylin in which the mordant is phosphotungstic acid

Lead Hematoxylin

demo of granules of endocrine cells of alimentary tract

Molybdenum Hematoxylin

demo of collagen, reticulin, and argentaffin cells

Cochineal dyes

Extracted from bug treated with alum to produce carmine.

Picro carmine

Cochineal dye with Picric acid used for neuropathological studies.

Best carmine

Cochineal dye with Aluminum Chloride used for Glycogen

Orcein

Vegetable dye extracted from lichens, used for staining elastic fibers.

Synthetic Dyes

AKA Coal Tar Dyes/ Aniline Dyes: Artificial Dyes.

Chromophore

Responsible for the coloring property of dye; Giving/imparting color

Auxochrome

Responsible for the dyeing property; Retaining the imparted color.

Lysochrome dyes / Oil Soluble dyes

used to demonstrate fats without auxochrome component.

Sudan Black B

Most sensitive of the oil soluble dye

Sudan IV

AKA Scharlach R - for neutral lipids, staining them red.

Sudan III

First sudan dye to be introduced into histochemistry; a good fat stain for CNS tissues.

Acid dye

cytoplasmic stain - Eosin - pink

Basic Dye

nuclear stain - Hematoxylin - blue/ bluish black

Neutral/Amphoteric Dye

Can stain both the nucleus and cytoplasm - Wright’s stain; Romanowsky Dyes

Autotechnicon

Regarded as an automatic tissue processor, can only do fixation up to infiltration.

Tissue Transfer Type / Dip and Dunk Type

• most common type.

• tissue is transferred from one reagent to another until the process is complete.

Fluid Transfer Type / Enclosed Types

• tissue being processed is within a chamber

• the fluid or reagent that is required in each process is pumped in and out of the chamber

Stains for Frozen Sections

• H & E stain

• Thionine

• Polychrome Methylene Blue

• Alcoholic Pinacyano

Basic solution

To remove excess acid dyes you will use what?

Acid solution

To remove excess basic dyes you will use what?