Immunohistochemistry

1. BIOL22332/20972 Genetics / Dev.Biol RSM MODULE 2 (Andreas Prokop) The University of Manchester Faculty of Life Sciences Immunohistochemistry

2. Drosophila developmental stages

3. 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.

4. What do experimental antibodies detect? • Where do experimental antibodies come from? • How does immuno-cyto/histochemistry work?

5. Differential gene expression- equipping cells with the right set of gene products UTR EX IN Gene non-coding transcription (epigenetics, TFs, differential start sites) pri- miRNAs primary transcript RNA processing (spliceosome, alternative splicing) mRNA miRNAs translation (ribosomes, initiation, elongation, termination factors) protein*** protein (2nd) folding, complex formation (chaperones) protein (tertiary, quaternary) posttranslational modifications (enzymes) protein*** (phosphorylation, glycosylation, ubiquitination...) degradation (proteasome, lysosome)

6. Detecting gene products at different levels Gene non-coding transcription (epigenetics, TFs, differential start sites) pri- miRNAs in situ hybridisation with intron probes primary transcript RNA processing (spliceosome, alternative splicing) in situ hybridisation with exon probes mRNA miRNAs translation (ribosomes, initiation, elongation, termination factors) protein*** protein (2nd) immunochemistry folding, complex formation (chaperones) protein (tertiary, quaternary) immunochemistry posttranslational modifications (enzymes) immunochemistry mass spectrometry protein*** (phosphorylation, glycosylation, ubiquitination...) degradation (proteasome, lysosome)

7. What do experimental antibodies detect? • Where do experimental antibodies come from? • How does immuno-cyto/histochemistry work?

8. Immune response

9. Antibodies http://www.news-medical.net/health/Antibody-Function.aspx

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

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

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

13. ABC enhancement kit diaminobenzidine (DAB)

14. What do experimental antibodies detect? • Where do experimental antibodies come from? • How does immuno-cyto/histochemistry work?

15. Summary of procedure - fixation (PF) - detergent (PBT) - 1st antibody - wash (PBT) - 2nd antibody - wash (PBT) - ABC kit - wash (PBT) - DAB/H202

17. Remove chorion: Images: Christoph Rickert

18. into the sieve: pour the bleach with the eggs into the sieve and wash with water Images: Christoph Rickert

19. Transfer into fix: transfer eggs with a brush Images: Christoph Rickert

20. 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

21. Incubate in fix for 30 min: Images: Christoph Rickert

22. Fixation Schiff base: ketimine, condensation product of a carbonyl group of an aldehyde or keton with an amino group 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)

23. remove PBS/fix (lower phase): Images: Christoph Rickert

24. Add methanol: Add 700µl of methanol Shake vigorously for 20 seconds!! Images: Christoph Rickert

25. After embryos sunk to the bottom: • 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

27. 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

28. 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. a) PAGENUMBER 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