The University of Manchester Faculty of Life Sciences Andreas Prokop BIOL20332/20972 GENETICS / Dev. Biol. RSM MODULE 2 Immuno histochemistry

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 (2 nd ) primary transcript 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...) UTRINEX protein (tertiary, quaternary) posttranslational modifications (enzymes) protein*** pri- miRNAs non-coding

What do experimental antibodies detect? Where do experimental antibodies come from? How to perform the experiment!

Immune response

Antibodies

How to obtain antibodies monoclonal antibodies injecting antigen X cells producing anti-X antibody producing hybridoma cells polyclonal antibodies injecting antigen X 1st boost 2nd boost bleed + purification

Use of secondary antibodies Y Y Y Y Y Y mouse antibody donkey anti-mouse Y Y Y Y Y Y rabbit antibody donkey anti-rabbit

Making antibodies visible crisp + permanent, double-labelling less optimal crisp, excellent double-labelling, but not permanent incremental but diffuse; mostly in situ hybridisation only EM, usually loss of tissue contrast (due to detergent)

ABC enhancement kit diaminobenzidine (DAB)

What do experimental antibodies detect? Where do experimental antibodies come from? How to perform the experiment!

- fixation (PF) - detergent (PBT) - 1 st antibody - wash (PBT) - 2 nd antibody - wash (PBT) - ABC kit - wash (PBT) - DAB/H Summary of the procedure

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:

mutant gene 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 7-9 groups C (x14) groups 13-15, 22 groups C-lacZ (x7) groups 19-21, 31 wt (x16) groups groups 27-29,32 aliquots (x20)(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 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 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.