Embryo fixation

1
BIOL22332/20972 Genetics / Dev.Biol RSM MODULE 3
The University of Manchester
Embryo fixation
Faculty of Life Sciences
2
(No Transcript)
3
EXPERIMENTAL OBJECTIVE of week 2 The aim of
this experiment is to describe nervous system
phenotypes in a set of Drosophila embryos
carrying unnamed mutations and hypothesise their
potential function and interrelation.
4
SHORT GUIDE TO THE LABORATORY PROTOCOL
Consider that in a real laboratory situation you
will not have the time to write long texts and
explanations. Therefore, find economic ways that
are nevertheless understandable - not only to
you, but also to others.
a) PAGE NUMBER and DATE b) AIM OF EACH
EXPERIMENT c) Details about your experimental
objects. (GENOTYPE, AGE or DEVELOPMENTAL
STAGE) d) Details about MATERIALS/CHEMICALS (e.g.
fixatives, antibodies etc.). e) SINGLE STEPS OF
YOUR EXPERIMENTS f) SPECIAL OBSERVATIONS,
PROBLEMS, TIPS, TRICKS, EXPLANATIONS or THOUGHTS
g) REFERENCES TO EXTERNAL SOURCES (pages in
manual, location of specimens, existence of
documentation) h) OUTCOME at intermediate stages
and upon termination of the experiment
5
(No Transcript)
6
Remove chorion
Images Christoph Rickert
7
into the sieve
pour the bleach with the eggs into the sieve and
wash with water
Images Christoph Rickert
8
Transfer into fix
transfer eggs with a brush
Images Christoph Rickert
9
Prepare fixative

• Everybody prepares a microfuge tube with fixation
  solution BEFORE STARTING THE WHOLE PROCEDURE
• label the tube appropriately (group, genotype,
  antibody)

500µl 4 formaldehyde in PBS
Images Christoph Rickert
10
Incubate in fix for 30 min
Images Christoph Rickert
11
remove PBS/fix (lower phase)
Images Christoph Rickert
12
Add methanol
Add 700µl of methanol
Shake vigorously for 20 seconds!!
Images Christoph Rickert
13
After embryos sunk to the bottom

• Remove all liquid (but not embryos!!!) and fill
  up with methanol
• Remove methanol and wash again with fresh
  methanol
• Exchange methanol for PBT

14
mutant gene BP102 (mouse) anti-A (mouse) anti-C (mouse) anti-lacZ (rabbit) anti-Fas2 (mouse)
A groups A B C groups J K
B groups D E F
C groups G H I groups L M
C-lacZ groups N O groups P Q R groups S T U groups V W (X)
15
Each group gets different batches of egg lays of
the same genotype
batch labelled No 1 Sat 6pm - Sun 9am ? stored
12ºC batch labelled No 2 Sun 9am - 2pm ? stored
18ºC batch labelled No 3 Sun 2 - 7pm ? stored
20ºC, 12ºC since Mon 10am batch labelled No
4 Sun 7pm - Mon 10am ? stored 25ºC, 12ºC since
Mon 10am