Workshop 2 - Visualising Cell Strucutres

Question 1

methylene blue:

Answer 1

methylene blue is a positively charged dye that binds to negatively charged nuclei acids such as DNA and RNA - as a result the nucleus and cytoplasm become blue and more visible

Question 2

hematoxylin & eosin:

Answer 2

combination of two stains: hematoxylin which stains the nucleus purple and eosin which stains the extracellular matrix pink

hematoxylin is a positively charged stain that that binds to negatively charged nucleic acids such as DNA and RNA while eosin is positively charged and binds to proteins in the cytoplasm

Question 3

gram staining:

Answer 3

gram staining is used to classify bacteria giving either gram positive or gram negative results

this is based on the stain reacting with with peptidoglycan (a polysaccharide) in the cell walls of the bacteria
–Those with a thick layer are Gram-positive
–Those with a thin layer are Gram-negative
•The peptidoglycan layer prevents the crystal violet in the stain from washing out during the ethanol wash

Question 4

antibodies are made up of:

Answer 4

they are made up of a dimer of heavy chains with light chains bound by disulfide bonds

at the top tips of the heavy chain, there is an antigen specific binding site which recognises specific molecules

Question 5

multiplexing:

Answer 5

as we can raise different antibodies against different antigens in different species, we can stain for multiple proteins and get different fluorescent signals for each protein – useful for identifying different cells or even structures

Question 6

DAPI:

Answer 6

•DAPI (4′,6-diamidino-2-phenylindole) is a fluorescent stain that binds to A/T-rich regions of DNA

•As such, this means that it preferentially is localised only to the nucleus where DNA is

•Can be useful to determine the location of the nucleus in a cell

•Also useful to locate the cell under the microscope for focusing since some fluorescent signal against proteins can be difficult to detect out of focus