The University of Manchester Faculty of Life Sciences Andreas Prokop BIOL20332/20972 GENETICS / Dev. Biol. RSM MODULE 2 Immuno histochemistry
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Andreas Prokop
BIOL20332/20972
GENETICS / Dev. Biol. RSM
MODULE 2
Immunohistochemistry
Drosophila developmental stages
EXPERIMENTAL OBJECTIVE of week 2
To understand how molecular mechanisms of axonal guidance can be studied via genetic loss-of-function analysis combined with
immunohistochemistry. For this, you will study nervous system defects of selected Drosophila mutant embryos.
• What do experimental antibodies detect?
• Where do experimental antibodies come from?
• How to perform the experiment!
Differential gene expression - regulating genes at different levels
Gene
mRNA
protein (2nd)
primarytranscript
transcription (epigenetics, TFs, differential start sites)
RNA processing(spliceosome, alternative splicing)
translation (ribosomes, initiation, elongation, termination factors)
folding, complex formation(chaperones)
protein***(phosphorylation, glycosylation, ubiquitination...)
UTR INEX
protein (tertiary, quaternary)
posttranslationalmodifications (enzymes)
protein***
pri-miRNAs
miRNAs
non-coding
• What do experimental antibodies detect?
• Where do experimental antibodies come from?
• How to perform the experiment!
Immune response
http://www.news-medical.net/health/Antibody-Function.aspx
Antibodies
How to obtain antibodies
monoclonal antibodies
injecting antigen X
cells producinganti-X antibody
producinghybridoma
cells
polyclonal antibodies
injectingantigen X
1st boost
2nd boost
bleed + purification
Use of secondary antibodies
YYY
YYY
mouseantibody
donkeyanti-mouse
YYY
YYY
rabbitantibody
donkeyanti-rabbit
Making antibodies visible
crisp +permanent,
double-labelling less optimal
crisp, excellent double-labelling,
but notpermanent
incremental but diffuse; mostly in situ hybridisation
only EM, usually loss of tissue
contrast (due to detergent)
ABC enhancement kit
diaminobenzidine (DAB)
NH2
NH2NH2
NH2
• What do experimental antibodies detect?
• Where do experimental antibodies come from?
• How to perform the experiment!
- fixation (PF)- detergent (PBT)- 1st antibody- wash (PBT)- 2nd antibody- wash (PBT)- ABC kit- wash (PBT)- DAB/H202
Summary of the procedure
Remove chorion:
Remove chorion:
Images: Christoph Rickert
Transfer into the sieve:
pour the bleach with the eggs into
the sieve and wash with water
Images: Christoph Rickert
Transfer into fix:
transfer eggs with a brush
Images: Christoph Rickert
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
Incubate in fix for 30 min:
Images: Christoph Rickert
Schiff base: ketimine, condensation product of a carbonyl group of an aldehyde or keton with an amino group
Fixation
Apart from cross-linking agent, proteins can be denatured/fixed through different means:
• Acids (e.g. acetic acid)
• solvents (e.g. ethanol, methanol)
• heat (e.g. 1 min 60°C)
Remove PBS/fix (lower phase):
Images: Christoph Rickert
Add methanol:
Shake vigorously for 20 seconds!!
Add 700µl of methanol
Images: Christoph Rickert
• Remove all liquid (but not embryos!!!) and fill up with methanol
• Remove methanol and wash again with fresh methanol
• Exchange methanol for PBT
• Wash once more with PBT
• Add primary antibody
After embryos sunk to the bottom:
mutantgene
BP102(mouse)
anti-A(mouse)
anti-C(mouse)
anti-βGal(rabbit)
anti-Fas2(mouse)
A (x12)groups
1-3 groups
4-6
B (x12)groups
7-9groups 10-12
C (x14)groups
13-15, 22groups 16-18
C-lacZ (x7) groups
19-21, 31
wt (x16)groups 23-26
groups 27-29,32
aliquots (x20) (x8) (x8) (x7) (x18)
Distribution of fly stocks across the course
Each group gets different batches of egg lays of the same genotype:
batch labelled No 1Sat 6pm - Sun 9am stored 12ºC
batch labelled No 2Sun 9am - 2pm stored 18ºC
batch labelled No 3Sun 2 - 7pm stored 20ºC, 12ºC since Mon 10am
batch labelled No 4Sun 7pm - Mon 10am stored 25ºC, 12ºC since Mon 10am
Source of egg lays
a) PAGE NUMBER and DATE
b) AIM/RATIONALE OF 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 (note that clear reference to the manual may suffice to cover methods)
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
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.