week 1 easter Flashcards
Give adv and disadv of phase-contrast microscopy vs. fluorescence
PC Adv: Easy to prepare samples No need to use dyes/fluorescent tags Can look live Disadv: Can't distinguish individual proteins Fixed Fluorescence: Adv: Can see individual proteins and more detail in each cell Can see multiple proteins at once Disadv: no live info Harder to prepare Expensive Stains/antibodies not always available
How are drosophila embryos fixed and washed
Fix : methanol
Wash : PBS containing detergent
How do you block unspecific sites to which antibodies may bind?
Incubate with PBS-S containing BSA
What specific primary antibodies did we add?
One that binds centrosomal protein Cnn
One that binds microtubule protein alpha-tubulin
What are secondary antibodies?
They bind primary antibodies to conjugate them to a dye
What did we mount the final embryos in
A gylcerol-based mounting medium containing DAPI (binds DNA and emits blue light under UV) and an anti-fade agent
What are steps of preparing drosophila testes for live phase imaging?
1) Add PBS drops on dissection slide
2) Break the pupal case at the head end and remove the fly head
3) pull the fly out of the case
4) Grip fly between thorax and abdomen
5) Grip tip on abdomen and slowly pull forceps apart: testes are spiral shaped
What 3 structures can you see in drosophila testes?
1) Spermatocytes (large with large nuclei)
2) spermatids–> have phase-dark Nebenkern (2 stranded helical structure at the top of tail) and phase light nucleus
3) Sperm bundles
Draw round spermatids at the onion phase
(see notes for diagram)
How do you know if meiosis went wrong?
Not a 1:1 ratio of nuclei and nebenkern
