Workshop 2 - Visualising Cell Structures and Proteins Flashcards
methylene blue
positively charged dye that binds to negatively charged amino acids (such as DNA and RNA)
result of staining with methylene blue
nuclei and cytoplasm become blue and more visible
haematoxylin & Eosin use
one of the most commonly used tissue stains in histology
haematoxylin
positively charged dye that binds to negatively charged nucleic acids (such as DNA and RNA)
result of staining with haematoxylin and Eosin (2)
- Haematoxylin stains nuclei purple
- Eosin stains extracellular matrix and cytoplasm pink
Eosin
positively charged dye that binds to proteins in cytoplasm
use of gram stain
used to classify bacteria (either gram-positive or gram-negative)
what does gram stain react with? (2)
- peptidoglycan (polysaccharide) in cell walls of bacteria
- peptidoglycan layer prevents stain from washing out during ethanol wash
results of Gram stain (2)
- thick layer = gram-positive
- thin layer = gram-negative
antibody structure
dimer of heavy chains with light chains bound by disulfide bonds
where is the antigen specific binding site found on antibodies?
top tips of heavy chain
target molecules of antibodies
antigens
primary antibodies
specifically raised antibodies against specific antigens (changed antigen-specific variable region)
fluorophores
fluorescent compound that can be attached to antibodies
issue with attaching fluorophore to single primary/secondary antibody
signal weak and hard to detect
secondary antibodies
antibodies created against antibodies themselves
how must secondary antibodies be raised
in different species to prevent cross-reactivity
purpose of multiple secondary antibodies with fluorophores binding to same primary antibody
amplification of signal (one antigen - one primary antibody - multiple secondary antibodies = multiple fluorophores)
what kind of antibody is: mouse anti-ß-actin primary antibody (naming)
primary antibody that will be bound to ß-actin, made in a mouse
what kind of antibody is: rabbit anti-mouse secondary antibody (naming)
secondary antibody that will bind to any mouse antibody, made in a rabbit
use of DAPI
useful to determine the location of the nucleus in a cell
DAPI
fluorescent stain that binds to A/T-rich regions of DNA
why is DAPI useful for locating cells under microscope instead of antibodies?
some fluorescent signals against proteins difficult to detect or focus
