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Stains
The only way to render the tissue visible under the light microscope is to provide color to the cell components through
Differential staining
This enables microscopic evaluation of all areas of the tissue.
hematoxylin and eosin (H&E) method.
most common "routine” staining
"Special stains"
Other alternative staining techniques employed when the H& E cannot visualize certain tissue components under consideration.
Hematoxylin
This stain has a deep blue-purple color and stains nucleic acids.
Incompletely understool
Hematoxylin stains by a complex, ____ ____ reaction
Eosin
stain that is pink and stains proteins non-specifically.
blue; pink
In a typical H&E stained section, nuclei appear _____ while the cytoplasm and extracellular matrix show different shades of _____.
Nuclear chromatin activity
this influences the varying depth of hematoxylin stain.
eosin; blue-tinged
Nucleoli stain with _____. Ribosome-rich cytoplasm appears _______
perinuclear absent
A prominent Golgi zone shows _______ ______ staining.
complete removal of paraffin
Staining always begins with the _________ in fixed sections on slides for the water-soluble stain solutions to act on the tissue.
dry storage; wet; coverslip
Mounted tissue samples are stable in ______ until the wax is removed; after which, the tissue must remain ______ until staining ends and the slide is mounted with a ______.
60° C; 30 minutes
Dewaxing starts with heating the slides in ______ oven for at least _____ to soften the wax.
couple; xylene
The slides are then immersed in a _____ of changes of clean _____
descending; distilled water
The slides are taken through _____ concentrations of alcohol to _______.
Lipid stain that absorb the fat
Example of pure physical stain
Chemical stain
Most stains fall under the this type of stain, in which the dye is adsorbed by the cell components.
van der Waals
ionic or electrostatic
covalent
hydrogen
It is then bound to the tissue through ______ forces, _______ or ______ bonds, ______ bonds, or _____ bonds.
basophilic
Tissue elements attracting basic dyes are
acidophilic
Those attracting acidic dyes are
Acidic
Anionic
Negatively charged
Nuclei are generally 1) 2) 3)
Basic
Cationic
Positively charged
Nuclei stain with ___ dyes
Dye-salt
Nuclei stains by forming _____ unions
Basophilic
The reaction in nuclear staining is termed
dyes-metal mordants
Nuclei that are positively charged, decalcified, or contain only basic nucleoproteins stain with _______ combination.
chemical forces
a complex interplay of _______operates on the second mechanism of nuclear staining
amino acid side chains
Cytoplasmic staining is brought about by proteins or charged groups on the _________ of proteins.
amphoteric; pH
Proteins are ______ because they can have either a net positive or negative ionic charge depending on the ____ of the solution.
isoelectric point ; pH 6
The _____ of proteins, in which the positive and negative charges are equal and with no electrical field migration, is approximately _____.
positive; anionic or negatively charged; eosin
In a solution below the IEP or below pH 6, the net charge on the cytoplasmic proteins is ______ and the affinity is for an ______ or _______ dye such as ____.
negative; cationic dye
Above the IEP or above pH 6, the net charge is _____ and the attraction is for a ______.
too low
If the pH of the solution is _____, eosin is uncharged and stains the tissue non-specifically
net negative
Above pH 6, proteins will have a _____ charge and will no longer attract eosin.
carmine
orcein
saffron
hematoxylin.
Most important natural dyes
organic
coal tar
benzene
All dyes are _____ compounds, mostly derivatives of _____ or _____.
A group that confers the property of color is called a chromophore, which is the basis for the general classification of dyes. A compound containing chromophoric groups is called a chromogen.
Chromogens
These substances may or may not be able to act as a dye or combine with cell components to be colored.
auxochrome
To enable the dye to link' to the tissue, an ionizing group called an ____ is required.
Amino group (NH2)
The auxochrome in the 'basic' dyes is the
aniline
_____ contains amino group, and many of the dyes are ____ derivatives.
sulfonic acid (SO2OH) group
The usual auxochrome in the 'acid' dyes is the ______, ionized to produce negative charges;
Carboxyl (COOH) groups
Hydroxyl (OH) groups
Two other acidic auxochromes are the ____ and ____ groups
positive charge
Basic dyes, properly called cationic dyes, are those dyes with a ______
negative charge
Acid dyes, or anionic dyes, are those with a _____.
chloride salts
Cationic dyes most frequently are
crystal violet
safranin
Examples of cationic dyes
sodium salts
anionic dyes most frequently are ___
orange G
picric acid
Example of anionic dyes
pH 3 to 9
Most dyes remain cationic or anionic throughout the pH range in which most staining occurs
cationic
Some dyes are amphoteric; they are ____ at pH below the IEP
Anionic
Some dyes are amphoteric; they are ____ at pH above the IEP
Hematein; 6.6
It is an example of an amphoteric dye having an IEP of about pH _____
Lipid stains
These are non-ionic compounds incapable of electrolytic dissociation.
False
Lipids are dyes, as they have been classified as neutral dyes
organic solvents; water
Lipid dyes are soluble in some ______ but not in ____
dissolving
Lipid stains 'color' certain tissue elements by _____ in them (physical staining).
pH of the solution
The tissue element and the dye molecule must have complementary charges in order to bind.
Increase in temperature
This swells the tissue and facilitates diffusion of dye molecules for effective dye action and rapid staining time.
Increased concentration of dye molecules
The difference in gradient propels tissue-dye binding.
Salt ions
_____ in the solution and dye ions probably compete for the same binding sites, altering the intensity of dye binding.
Presence of other salts
competition for the same binding sites, altering the intensity of dye binding.
type of fixative
the ____ alters the staining character of the tissue by reacting with certain chemical groups in the tissue and making them unavailable for dye binding,
Formalin
____ reacts with the tissue amino (NH2 group)the primary eosin binder, lessening the eosin tissue uptake.
potassium dichromate
This fixatives take up less hematoxylin than eosin
Zenker
Bouin
unbuffered formalin
A tissue overexposed to acidic fixatives such as ___, ___, or ____ will lose its basophilic (nuclear) staining properties
Progressive staining
it is a staining method in which the reaction proceeds 'forward and is stopped once the desired intensity of color is achieved.
Regressive staining
the tissue is overstained and then differentiated and then decolorized, until only the desired element remains stained
mordant
This is substance is usually a metal, that links the dye to the tissue.
Basic
Mordant combines with the dye to form a "lake" that is usually _____ in action.
progressively
Many dyes, including counterstains (cytoplasmic stains) and even mordant dyes, are used ____.
Regressively
Mordant dyes are more effective when used _____ to achieve differential staining
weak acid
Basic (cationic) dyes are differentiated by ____ solutions
weak alkaline
acidic (anionic) dyes are differentiated by ____ solutions.
Aluminum hematoxylins
dilute HCl solution
Eosin
Dilute ammonium hydroxide solution
Alcohol
If the differentiating solutions are prepared in ____ instead of water, better control of decolorization is possible.
tissue-mordant dye
Excess mordant in the differentiating solution will dissolve the ______
True
The most dye-bound tissue elements will decolorize last.
Oxidizing differentiators
are substances added to the dye that oxidize it to a colorless substance, in effect differentiating it as well.
Potassium permanganate
this chemical is used as an oxidizing differentiator
Hematoxylin
most widely used nuclear stain
Haematoxylon campechiamum
Hematoxylin is extracted naturally from the log wood called ___ although synthetic equivalents are available
True
Hematoxylin per se is not a dye
hematein; anionine
it is the oxidation product of hematoxylin, is a weak _____ dye.
ripening
In the process termed _____, oxidation is achieved either by exposing the solution to air or by mixing oxidizers.
false
Oxidation to hematein is not needed.
pH
Alkaline
acidic
The oxidation rate is ______ dependent, as hematein forms rapidly in ____ solutions but slowly in ____solutions.
sodium iodate
potassium permanganate.
Oxidizers mixed with haematein to oxidize
Harris hematoxylin
This is the hematoxylin commonly used in routine H&E
Periodic Acid-Schiff (PAS)
selected special stains such where harris hematoxylin is used
mercuric oxide
sodium iodate
There are ready-to-use preparations of harris hematoxylin available in the market. The original formula uses _____ (toxic) but is now replaced by ______.
hematoxylin
Absolute ethyl alcohol
Harris hematoxylin preparation:
Dissolve ____ in ____
ammonium aluminum sulfate
Distilled water
Harris hematoxylin preparation:
dissolve _____ in ______ with heat.
Sodium iodate (oxidizer)
Harris hematoxylin preparation:
Mix the 2 solutions and bring rapidly to a boil. Remove it from heat and slowly add_____.
Dark purple
Harris hematoxylin preparation:
Reheat it to boiling until the solution becomes ___ ____. Remove it from heat, and immediately place the vessel into a basin of ice.
metallic sheen
The harris hematoxylin stain is ready for use after cooling or when there is a _______ on the surface of the solution.
glacial acetic acid
4mL
96 mL
Filter the harris hematoxylin before use, and add ______ to give a final concentration of 4% (_____ of glacial acetic acid for every _____ of hematoxylin).
progressive stain
Regressive stain
Harris solution is used best as a _____(as originally intended), but may also be used as a _______.
1g
How many grams of hematoxylin is present in mayer hematoxylin
1000mL
How many mL of distilled water is present in mayer hematoxylin