Immunohistochemistry

BIOL22332/20972

Genetics / Dev.Biol RSM

MODULE 2 (Andreas Prokop)

The

Uni

vers

ityof

Man

ches

ter

Fac

ulty

of

Life

Sci

ence

s

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.