Staining Flashcards
Affinity of nucleus to stains
Acidic (nucleus) »_space;»»> basic stains
The process of applying dyes on the sections to study architectural pattern of the tissue and physical characteristics of the cells.
Staining
Affinity of cytoplasm to stains
Basic (cytoplasm)»_space;»»> acidic stains
MAJOR GROUPS OF TISSUE STAINING
- HISTOLOGICAL STAINING
- HISTOCHEMICAL STAIN (HISTOCHEMISTRY)
- IMMUNOHISTOCHEMICAL STAINING
The process whereby the tissue constituents are demonstrated in sections by direct interaction with a dye or staining solution
HISTOLOGICAL STAINING
Examples of Histological staining
micro-anatomical stains, bacterial stains, specific tissue stains (e.g. muscles, connective tissue and neurologic stains)
The process whereby various constituents of tissues are studied thru chemical reactions that permits microscopic localization of specific tissue substances
Histochemical staining (Histochemistry)
Examples of Histochemical staining
Perl’s prussian blue reaction for hemoglobin and periodic acid schiff staining for carbohydrates
Enzyme histochemistry:
Active reagent?
Tissue?
Active reagent: substrate
Tissue: enzymes
A combination of immunologic and histochemical techniques that allow phenotypic markers to be detected by antibodies (e. g. polyclonal, monoclonal, enzyme- labeled or fluorescent-labeled)
IMMUNOHISTOCHEMICAL STAINING
Methods of Staining
- Direct Staining
2. Indirect Staining
Uses aqueous or alcoholic dye solutions (e.g. methylene blue, eosin) to produce a color
Direct Staining
Uses a mordant or another agent to intensify the action of the dye used
Indirect Staining
Serves as a link or bridge between the tissue and the dye
Mordant
The dye may stain weakly by itself, therefore the mordant combines with the dye forming a colored “lake” which would combine with the tissue forming an insoluble “tissue-mordant-dye-complex”, which would allow subsequent counterstaining and dehydration
Mordant
Examples of mordant
Potassium alum with hematoxylin in Ehrlich’s hematoxylin
Iron in Weigert’s hematoxylin
- Not essential and does not participate to the chemical reaction of the tissue and dye
- Accelerates the speed of the staining reaction by increasing the staining power and selectivity of the dye
Accentuator
Examples of Accentuator
Potassium hydroxide in Loeffler’s methylene blue, Phenol in Carbol thionine and Carbol fuchsin
- Tissue elements are stained in definite sequence
- The staining with specific periods of time or until desired color is attained
Progressive Staining
-First over-stain the tissue to obliterate cellular details
-Excess stain is removed or decolorized from unwanted parts of the tissue and until the desired color is obtained
Regressive Staining
The selective removal of excess stain from the tissue during regressive staining so that a specific subatance may stain distinctly from the surrounding tissue
DIFFERENTIATION / DECOLORIZATION
Differentiation/decolorization is usually done by washing the section in?
in simple solution (e.g. water or alcohol) or use of acids and oxidizing agents
Differentiator for both acidic and basic dyes by dissolving excess dye
Alcohol
Can oxidize hematoxylin to a soluble, colorless compound.
Disadvantage: if a mordant stained section is allowed to remain in a differentiating agent such as 1% or 2% alcohol, all of the dye will be removed.
– Restaining faded slides
Mordant (e.g. iron alum)
Makes use of specific dyes which differentiate particular substances by staining it with a color that is different from that of the stain itself (metachromasia)
Metachromatic Staining
Metachromatic dyes (basic) belongs to?
Thizine and triphenylmethane groups
Examples of metachromatic dyes
- Methyl violet or crystal violet
- Cresyl blue (for reticulocytes)
- Safranin
- Bismarck brown
- Basic fuchsin
- Methylene blue
- Thionine
- Toluidine blue
- Azure A, B, C
Necessary for most metachromatic staining techniques
Water
Application of a different color or stain to provide contrast and background to the staining of the structural components to be demonstrated.
COUNTERSTAINING
Cytoplasmic stains:
Red?
Yellow?
Green?
Red: Eosin Y, Eosin B, Phloxine B
Yellow: Picric acid, Orange G, Rose Bengal
Green: Lt. Green SF, Lissamine Green
Nuclear Stains:
Red?
Blue?
Red: Neutral Red, Safranin O, Carmine, Hematoxylin
Blue: Methylene blue, Toluidine Blue, Celestine blue
The process where specific tissue elements are demonstrated not by stains but by colorless solutions of metallic salts which are deposited on the surface of the tissue
Metallic Impregnation
Solutions used for metallic impregnation
Gold chloride, Silver nitrate
The selective staining of living cell constituents
Nucleus is resistant to this type of staining
Vital Staining
Vital Stains demonstrates cytoplasmic structures by
By engulfment of the dye particle
By staining of pre-existing cellular components
Two types of vital staining
- Intravital staining
2. Supravital staining
Staining by injecting the dye into any part of the animal body
-e.g. lithium, carmine and India ink
Intravital staining
Used immediately after removal of cells from the living body
e.g. Neutral red (best), Janus green (mitochondria), Trypan blue, Nile blue, Thionine and Toluidine Blue
Supravital staining
Most common method utilized for microanatomica studies of tissues
Routine Hematoxylin and Eosin (H & E)
H & E result:
Nuclei
Blue to blue black
H & E result:
Karyosome
Dark blue
H & E result:
Cytoplasm
Pale pink
H & E result:
RBCs, eosinophilic granules, keratin
Bright-orange red
H & E result:
Calcium and decalcified bone
Purplish blue
Decalcified bone matrix, collagen, osteoid
Pink
