G-banding Flashcards
1
Q
Why are Superfrost slides used?
A
- high quality microscope slides suitable for a range of cytogenetic techniques and cells will adhere to these with no additional chemical agents
2
Q
How are slides washed?
A
- soak o/n in 5% Decon90
- rinse in 5 changes of water
- indiv wash in distilled water
store in beaker of distilled water at 4° covered w/ clingfilm until use
3
Q
Why is height of slide dropping important?
A
- critical to ensure the proper spreading of chromosomes w/o causing damage to the cell
- too low = poor spreading of chromosomes
- too high = cells can burst
4
Q
What is the importance of slide drying?
A
- as fixed cell hits surface of wet slide, it sits in a solution containing fixative and water
- fixative is hygroscopic (= attracts water from the surrounding environment) and therefore intracellular fixative causes small influx of water into fixed cell, increasing its vol and distance between individual chromosomes further
- as liquid on slide evaporates, the migrating meniscus exerts downward
pressure that causes stretching of the plasma membrane and flattening of cell - plasma membrane is thought to be more flexible in fixed cells due to swelling and exposure to fixative
- flattening process continues as slide dries and causes cell membrane to
become thinner, cell to occupy greater area on slide surface and chromosomes to spread correctly - Superfrost slides must be stored in cold water to ensure slow, constant
drying
5
Q
What happens if slide dries too quick or too fast?
A
- too quickly = poor spreading (encapsulation) can occur
- too slowly = cell can burst due to prolonged stretching of plasma membrane
6
Q
What is encapsulation?
A
- metaphases poorly spread, extensive chromosomal overlap is present and high background/low resolution makes single chromosome analysis difficult
- cytoplasmic components gen background signals by absorbing light passing t/ cell when performing light microscopy, worse in encapsulated cells as cytoplasmic layer much thicker so much higher background observed as a ‘haze’
7
Q
How are slides aged?
A
- exposed to sunlight at room temperature for 48 hours
- purpose is to denature proteins, remove residual fixative, enhance adherence to the glass and remove water from the chromosomes which significantly improves the quality
8
Q
What is Leishman’s staining?
A
- methylene blue based chemical stain that stains chromatin blue
- also used when cells/ metaphases need to be quickly stained to check eg. total chromosome no. immediately after dropping a slide and is often referred to as block staining
9
Q
How does slide mounting work? Why is it needed?
A
- eg. DPX
- sits between sample and coverslip
- most mounting medias undergo a polymerisation/curing process when exposed to air and turn from a liquid to semisolid state, minimising movement in sample
- essential for high-magnification oil-immersion microscopy
- preserves processed sample for long periods of time
10
Q
How does trypsin produce banding pattern?
A
- degrades histones in chromatin nucleosomes and causes local chromatin
structure to collapse - large prop of histones in highly condensed heterochromatic regions shielded from exposure to trypsin and chromatin structure maintained
- in euchromatic regions the open chromatin conformation allows trypsin access to histones and greater disruption of chromatin structure
- so following trypsin treatment and Leishman’s staining euchromatin regions stain lightly and heterochromatic regions exhibit dark staining, as their chromatin structure more intact and able to bind more Leishman’s
11
Q
What factors affect G-banding quality?
A
- time exposed to trypsin
- conc of trypsin
- temp of trypsin
