1. Immunolabeling of Cells and Transmission Electron Microscopy Ike Miguel June 30, 2006 ABE Workshop 2006

2. Purpose Using TEM to label specific sites in Arabidopsis thaliana. Goal Overview of Immunolabeling Preparation of samples and viewing Observations of results Thoughts

3. Antibodies are used in immunolabeling of cells Immunoglobulins, or antibodies, detect foreign bodies and attach at specific sites called epitopes Antibodies fused to myeloma cells where they are cloned indefinitely and screened in vitro and in vivo Indirect method used to our purposes

4. Antibody Structure 4 polypeptide chains Two light, VL and CL Two light, VL and CL 2 heavy chains determine fuctional activity in vivo IgG, IgA, IgM, IgE, IgD IgG, IgA, IgM, IgE, IgD

5. Use of Antibodies for Immunolabeling High level of specificity needed due to non- covalent, weak bonding Eliminate non-specific binding Optimal conditions include reagants, pH, heat. Controls are very important! Negative controls to discriminate non-specific binding Negative controls to discriminate non-specific binding

6. Tranmission Electron Microscopy Zeiss 10/A conventional TEM Easy operation Easy operation Uses film for images Uses film for images LEO 912 Energy-Filtering TEMLEO 912 Energy-Filtering TEM Little as 0.5um sectionsLittle as 0.5um sections EELS, ESIEELS, ESI Digital imagesDigital images Ultramicrotomy, 60-90nm sectioning in resinUltramicrotomy, 60-90nm sectioning in resin

7. Immunolabeling with Gold by Indirect Method Gold small, round and dense Easily detectable 1:10 and 1:100 colliodal gold-labeled conjugated to primary antibody

8. Preparation of Specimens by Embedding Preparation is limiting factor! Chemical fixation with aldehydes which cross-links proteins Paraformaldehyde for grids Paraformaldehyde for gridsDehydrate Infiltrate with resin Polymerize May stain prior to polymerization May stain prior to polymerization 7 – 10 days to fix. May need to repeat.

9. Ultramicrotomy and transfer to grids Glass knife can cut ucron thick sections Diamond knife can cut as small as 2-3um Our samples 80nm – 1um thick 60-80nm max thickness for transmission of light 2 sections “embedded” per grid Avg of 6 sections per grid (2 – 20) Avg of 6 sections per grid (2 – 20)

10. Post-Embedding and Labeling of Specimens Dilution series and controls! Sectioned on Formvar coated grids Pretreat and etch with Na-metaperiodate/ PBS-glycine Wash with TBST (detergent, ampiphatic, nonionic) Block with milk (lipid, naturally sticky) 1 o antibody Wash with TBST 2 o antibody, colliodal-gold Wash with TBS and H 2 0 to rinse any Ppt

11. TEM – LEO912 Energy-Filtering TEM Tungsten cathode used to produce electron illumination source Nitrogen for carbon residue Accelerated through column EMF used to bend and focus illumination EMF used to bend and focus illumination Grid sample on objective lense Projector lense produces visible light Beam strikes CCD strip for histogram results

12. GFP, DTT treated Grid 1: No pretreatment, 1:10 anti-GFP (1 o -Ab), 1:10 gold (2 o -Ab) DTT – dithiothritol used to denature GFP Expected labeling on ER, vacuole, or vesicles. What is this? Fig. 1 Numerous binding to organelle. Group 1.

13. GFP DTT non-specific binding Grid 2: Glycine, 1:10 anti-GFP (1 o -Ab), 1:10 gold (2 o -Ab) labeling Non-specific binding is expected result Thoughts? Vascular system or chloroplast Fig. 2 Non-specific binding

14. Group 2 GFP no DTT treatment Grid 3. No pretreatment, 1:10 anti-GFP, 1:100 gold. Possible labeling on vacuole. Grid 4. Na-Periodate, 1:100 anti-GFP, 1:100 gold. Random labeling

15. PDI2 Expected labeling on starch granules No pretreatment At high concentrations primary antibody may have a large number of non-specific binding. Grid 1. No pretreatment, 1:10 anti-PDI-2, 1:10 gold. Observing non-specific binding. Group 3

16. Group 3 PDI2 Grid 1. A lot of non-specific binding but closer observations may give insight.

17. Group 4 PDI2 Grid 4. Na-Periodate, 1:10 anti-PDI2, 1:1000? gold. Labeling of starch granules

18. Group 4 Control Grid 4. Control with no primary antibody.

19. Group 4 CNGC Cyclic nucleotide gated channel Allows passage of K+ Expected labeling on plasma membrane Grid 2. Glycine, 1:10 anti-CNGC, 1:10 gold

20. Group 4 CNGC Grid 2. Gycine, 1:10 anti-CNGC, 1:10 gold. Possible communication.

21. CNGC Group 4 Grid 3. No pretreatment, 1:10 anti-CNGC, 1:100 gold. Group 4

22. Errors and Conclusion Preparation is the limiting factor! Gold labeling antibody may have been prepared in the wrong concentrations. A set of new dilutions may be needed.