immunohistochemistry biol22332/20972 genetics / dev.biol rsm module 2 (andreas prokop) the...
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Immunohistochemistry
BIOL22332/20972
Genetics / Dev.Biol RSM
MODULE 2 (Andreas Prokop)
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Drosophila developmental stages
Immunohistochemical analyses performed during this course
EXPERIMENTAL OBJECTIVE of week 2:
The aim of this experiment is to describe nervous system defects of selected Drosophila mutant embryos, in order to illustrate molecular mechanisms that regulate axonal CNS midline crossing - and the experimental use of genetic
loss-of-function analysis and immunohistochemistry.
• What do experimental antibodies detect?
• Where do experimental antibodies come from?
• How does immuno-cyto/histochemistry work?
Differential gene expression - equipping cells with the right set of gene products
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)
pri-miRNAs
non-coding
miRNAs
protein***
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...)
protein (tertiary, quaternary)
posttranslationalmodifications (enzymes)
pri-miRNAs
non-coding
miRNAs
protein***
in situ hybridisationwith exon probes
in situ hybridisationwith intron probes
immunochemistry
immunochemistrymass spectrometry
immunochemistry
Detecting gene products at different levels
• What do experimental antibodies detect?
• Where do experimental antibodies come from?
• How does immuno-cyto/histochemistry work?
Immune response
http://www.news-medical.net/health/Antibody-Function.aspx
Antibodies
How to obtain antibodies
polyclonal antibodies
injectingantigen X
injecting antigen X
cells producinganti-X antibody
producinghybridoma
cells
monoclonal antibodies
1st boost
2nd boost
bleed + purification
Use of secondary antibodies
YYY YYYYYY YYY
rabbitantibody
mouseantibody
donkeyanti-mouse
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
NH2
NH2NH2
NH2
diaminobenzidine (DAB)
• What do experimental antibodies detect?
• Where do experimental antibodies come from?
• How does immuno-cyto/histochemistry work?
- fixation (PF)- detergent (PBT)- 1st antibody- wash (PBT)- 2nd antibody- wash (PBT)- ABC kit- wash (PBT)- DAB/H202
Summary of procedure
Remove chorion:
Images: Christoph Rickert
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
• Add primary antibody
After embryos sunk to the bottom:
mutantgene
BP102(mouse)
anti-A(mouse)
anti-C(mouse)
anti-lacZ(rabbit)
anti-Fas2(mouse)
Agroups A B C
groups J K
Bgroups D E F
Cgroups G H I
groups L M
C-lacZgroups
N Ogroups P Q R
groups S T U
groups V W (X)
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
Each group gets different batches of egg lays of the same genotype:
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 (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.