PRINCIPLES OF STAINING Flashcards
• The process of applying dyes on the sections
STAINING
is the process whereby tissue components are made visible in microscopic sections by direct interaction with a dye or staining solution
Staining
Staining
To see and study the_____ of the tissue, the _______of the cells, and the ______of the tissue and their cells
architectural pattern
physical characteristics
structural relationship
Staining
Made possible because of:______ of tissue and cells for dyes
Varying affinity
Most cells are______ and______, and therefore histological sections have to be stained in some way to make the cells visible. The same is true of components of the extracellular matrix.
colorless and transparent
A_______ is the purified form of a coloring agent or crude dye that is generally applied in an aqueous solution
histologic stain
Certain parts of cells and tissues that are acidic in character (e.g. nucleus) have greater affinity for______ dyes, while basic constituents (e.g. cytoplasm) take more of the____ stains.
Individual variation of the tissue constituents regarding these properties will consequently produce variation in colors under the microscope.
basic
acid
Many dyes, however, require the use of a________ a chemical compound that reacts with the stain to form an insoluble, colored precipitate on the tissue and make the staining reaction possible.
When excess dye solution is washed away, the_____ stain remains
mordant
mordanted
The great majority of routine histology is done with_________, because it is quick, cheap and informative.
It involves the use of two contrasting stains, e.g.,______ which stains the nuclear detail, and_____ which brings out the cytoplasmic detail of the cell and the tissue’s architecture.
hematoxylin and eosin (H&E) staining
hematoxylin
eosin
_______is poorly permeable to most staining solutions and should therefore be removed from the section prior to staining.
This is usually done by immersing the section in a solvent (e.g.______) two times, at 1-2 minutes duration each, for sections up to____ micron thick.
Paraffin wax
xylene
10um
is not miscible with aqueous solutions and low graded alcohol, and should therefore be subsequently removed with absolute alcohol, followed by descending grades of alcohol to prevent damage and detachment of sections.
Xylene
The______ is then finally replaced with water before actual staining of section is performed.
Such procedure is the exact reverse of impregnation and may be summed up by the phrase “Sections to Water”.
alcohol
If drying is not complete, the section (or part of it), especially from_____ and_____ tissue, may become detached from the slide during the process of staining, usually after adding the acid differentiator.
bone and nervous
If an_____ is to be used, there is no more need to replace the alcohol with water
alcoholic stain
After staining, the section is again______ with increasing grades of alcohol and cleared with two changes of_____ to prepare the section for mounting, since most mountants are miscible in _____.
dehydrated
xylene
True or False
The stained section may be left in xylene for an indefinite period of time until it is finally mounted on the slide.
True
True or False
The section should not be allowed to stay in alcohol for a Jong time because many stains are usually removed by prolonged immersion in alcohol.
True
Sections must be left in the oven for a minimum of_____ minutes before they are finally stained to avoid such problems.
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CLASSIFICATION OF STAINING
According to purpose
Histological
Histochemical
Immunohistochemical
is the process whereby the tissue constituents and general relationship between cell and tissue are demonstrated in sections by direct interaction with a dye or staining solution, producing coloration of the active tissue component.
Histological staining
Micro-anatomic stains, bacterial stains and specific tissue stains (e.g. muscles, connective tissue and neurologic stains) fall into this category.
Histological staining
METHODS OF STAINING (10)
- Direct Staining
- Indirect Staining
- Progressive STaining
- Regressive Staining
- Microanatomical Staining
- Metachromatic Staining
- Counterstaining
- Metallic Impregnation
- Vital Staining
- Intravital Staining
is the process whereby various constituents of tissues are studied thru chemical reactions that will permit microscopic localization of a specific tissue substance.
Histochemical staining
Chemical ions such as calcium, molecules such as bile pigments, and biopolymers such as cellulose, DNA and specific enzymes are among the tissue components that can be identified using…
histochemical staining techniques
The staining techniques employed for ______ are also usually applied for staining of histologic structures.
Examples of such type of stains are_______ reaction for hemoglobin, and_______ staining for carbohydrates.
histochemistry
Perl’s Prussian blue
Periodic Acid Schiff
is a combination of immunologic and histochemical techniques using a wide range of polyclonal or monoclonal, fluorescent labeled or enzyme-labeled antibodies to detect and demonstrate tissue antigens (e.g., proteins) and phenotypic markers under the microscope
IMMUNOHISTOCHEMICAL (IHC) STAINING
is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors, in the localization of biomarkers and differentially expressed proteins in different parts of a biological tissue, and in the detection of specific molecular markers that are characteristic of particular cellular events such as proliferation or cell death (apoptosis).
Immunohistochemical staining
Visualizing an antibody-antigen interaction can be accomplished in a number of ways.
Immunohistochemical staining
are used to label defined antigens with monoclonal and polyclonal antibodies.
Immunohistochemical staining techniques
Commercially produced antibodies most frequently originate from____, and less frequently from rabbits.
mice
The current recommendation for immunohistochemical techniques is a maximum of______ solution and, for some antibodies, fixation time can be up to a maximum of____
Microwave-based fixation of tissue in formaldehyde may have an adverse effect on immunohistochemical staining.
4% neutral buffered formaldehyde
48 h
is the process of giving color to the sections by using aqueous or alcoholic dye solutions
Direct staining
In______ staining only one dye is used, which is washed away after 30–60 seconds, prior to drying and examination.
simple (or direct) staining
is a classic example of a simple stain. This blue stain will color all cells blue, making them stand out against the bright background of the light microscope
Methylene blue
is the process whereby the action of the dye is intensified by adding another agent or a_____ which serves as a link or bridge between the tissue and the dye, to make the staining reaction possible
Indirect staining
MORDANT
By itself, the dye may stain only weakly, if at all.
The mordant combines with a dye to form a colored “_____”, which in turn combines with the tissue to form a “______” that is rendered insoluble in ordinary aqueous and alcoholic solvents.
lake
tissue-mordant-dye-complex
Lake
Mordant + Dye