1. Genetics / Dev.Biol RSM Embryo fixation BIOL22332/20972 MODULE 3
The University
of Manchester
Life Sciences
Faculty of
Embryo fixation

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

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 eggs with a brush
Transfer into fix:
transfer eggs with a brush
Images: Christoph Rickert

9. Prepare fixative: 500µl 4% formaldehyde in PBS
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. Shake vigorously for 20 seconds!!
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. A B C C-lacZ groups A B C J K D E F G H I L M N O P Q R S T U V W (X)
mutant
gene
BP102
(mouse)
anti-A
anti-C
anti-lacZ
(rabbit)
anti-Fas2 A
groups
A B C
J K B
D E F C
G H I
L M
C-lacZ
N O
P Q R
S T U
V W (X)

15. Sat 6pm - Sun 9am  stored 12ºC batch labelled No 2
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