Transcript 1° ab - Figshare

The University
of Manchester
BIOL20332/20972
GENETICS / Dev. Biol.
RSM
Faculty of
Life Sciences
MODULE 2
Embryo staining,
embedding,
documentation
& filing
Andreas Prokop
Today's procedures
- fixation
- detergent
- 1° ab
- wash
- 2° ab
Today's procedures
- fixation
- detergent
- 1° ab
- wash
- 2° ab
- wash 15‘
- ABC 1hr
- wash 15‘
- DAB/H202
H2N
H2N
NH2
NH2
Today's procedures
• rinse with PBT in centrifuge tube (2 x for 3-5 min. each)
• in parallel prepare your dissecting scope (light from top, white side of bottom
plate)
• remove PBT and add H202/DAB solution (handed out) - You must wear
gloves!
• close centrifuge tube and shake, then quickly open and pour its content into a
glass well; take up some liquid from the glass well to wash out embryos that
got stuck in the tube
• observe staining progress under the dissecting microscope - stop on time to
avoid excessive background staining - ask course assistants
• remove the H202/DAB solution with a pulled-out Pasteur pipette into the
original 15ml Falcon tube and replace with PBT
• one more time rinse with PBT (dispose off in original 15ml Flacon tube DAB-contaminated liquid, washing solutions and tips will be collected in
yellow bags for incineration)
• by gently rotating the well gather embryos at bottom of well – transfer back
to centrifuge tube with Eppendorf pipette (blue tip)
• remove PBT from centrifuge tube and add 700µl of 90% glycerol
Today's procedures
•
in a microfuge at 3000 rpm for 2 min, spin embryos down for several
minutes; they will settle on one side of the tube
•
cut off the very tip of a yellow pipette tip with a razor blade, set the pipette
to 100 μl, and pick up as many embryos as possible into this volume; to
achieve this, place the pipette tip in the area of the tube wall with the highest
embryo density, then slowly release the button of your pipette moving the tip
gently forward.
•
repeat this process for each slide, and spin down embryos again, if they
dispersed away from the tube wall.
Embedding of larval preparations
- transfer embryos in a small drop of glycerol (70 µl)
- put on cover slip, then let more oil flow in from the fringe, if required
- seal all around the cover slip with nail varnish
Filing of slides
The formation of neuronal cicuits
Genes involved in axon growth
germ cell migration
http://flymove.uni-muenster.de/Organogenesis/Gonads/OrgGonMov/pole_cells_lat120.mov
http://flybase.org/.bin/fbimage?FBbt:00005093
Optimising microscope settings
Documentation
Documentation
Templates are provided
Fly stocks and antibodies by group
mutant
gene
BP102
(mouse)
anti-A
(mouse)
anti-C
(mouse)
A (x12)
groups
1-3
groups
4-5, 22
B (x12)
groups
7-9
groups
10-12
C (x14)
groups
13-15, 6
groups
16-18
anti-Fas2
(mouse)
groups
19-21, 31
C-lacZ (x7)
wt (x16)
aliquots
anti-βGal
(rabbit)
(x20)
groups
23-26
groups
27-29,32
(x8)
(x8)
(x7)
(x18)