The University of Manchester Faculty of Life Sciences Andreas Prokop BIOL20332/20972 GENETICS / Dev....

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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.