Tissue prep and staining Flashcards
what are the steps needed to prepare tissues for observation
fixing, dehydration, removal of alcohol, embedding
what does fixation prevent
further deterioration of the specimen and helps to harden the tissue prior to embedding and sectioning
what is the main downfall of fixation
it radically distorts the specimen
what is one of the most widely used fixing agents
Formalin
what does formalin do
fixing agent that reacts with the amino acids of the tissue proteins and stabilizes tissue structure to prevent further deterioration
what do acid fixatives fix? don’t fix?
fix: chromatin, nuclei and spindle fibers
don’t fix: mitochondria and nucleoplasm
name three acid fixatives
carnoy’s fluid
zenker’s fluid
Bouin’s fluid
what is Carnoy’s fluid good for
good general fixative and is useful for preserving glycogen in animal tissues
what is Zenker’s fluid good for
useful when sharp histological detail is desired, but must be washed out carefully to prevent the precipitation of black crystals
what is Bouin’s fluid good for
widely used general fixative that gives cytological detail
when are basic fixatives usually used
when mitochondrial staining is desired
what is the main downfall of basic fixatives
chromatin is dissolved
what are 2 main fixatives for TEM
Glutaraldehyde: preserves proteins by cross-linking them
Osmium tetroxide: reacts with lipids and imparts electron density to cell and tissue structure
why must dehydration occur for tissue fixing and embedding?
because the tissue sample will eventually be embedded and infiltrated with a hydrophobic material (usually paraffin)
what is clearing?
consists of replacing the alcohol with an agent such as xylene or cedar oil
what are the steps of embedding
- the tissue specimen is moved sequentially through several melted paraffin baths
- after the final bath the specimen is placed in a mold that is then filled with melted paraffin
- the paraffin mold is rapidly hardened by placing it in a cold water bath
steps to prepare for staining
- paraffin must be removed from the section, which is now mounted on a microscope slide (accomplished with xylene)
- xylene must be removed using a graded series of alcohol down to water
- stains are then applied and the section is again dehydrated through a graded series of alcohols
- the alcohol is removed with xylene
- a drop of cement followed by a cover slip is applied
what is hematoxylin derived from
logwood as hematein
what can orcein and resorcin fuchsin stains be used for?
to reveal elastic material
what is silver impregnation useful for
to show reticular fibers and basement membranes
how do acid dyes bind to tissue components?
by forming electrostatic linkages with cationic groups such as the amino groups of proteins
define metachromasia
phenomenon whereby a dye changes color after reacting with a tissue component
what can histochemical techniques be used for
to study the chemistry of cells and tissues
what does the schiff reaction depend on
formation of aldehyde groups following exposure to HCL or periodic acid
what is periodic acid used for
used to cleave bonds between adjacent carbons of carbohydrates and form aldehyde groups
what is best carmine used for
dye that may be used to demonstrate glycogen deposits
what can immunocytochemical techniques be used for
to study the presence of specific tissue constituents (antigens) by using monoclonal antibodies
what are monoclonal antibodies derived from
activated B cell clones exposed to a specific antigen
