Immunolabeling and Transmission Electron Microscopy

Our Samples

1) GFP-Control (GFP – 2SC)

2) GFP-Treated with Tunicamycin

3) Wild Type

4) Embryo(PDI2-GFP)

Goals?

• GFP protein gene is fused with 2SC gene which vacuole targeted gene.

• Treatment with tunicamycin – GFP will be folded, not glycocylated. Lot of unfolded protein in secretory appratus- cause traffic jam. =>GFP probably have a hard time to leave=> we expect see lot more GFP protein in endomembrane system.

GFP??

Green Fluorescent Protein?: It’s a protein.

238 amino acid sequences.

From Jelly fish “Aquorea

Victoria” Blue light hits

Green lights out.

Can be used as protein tracker?

Fixation

• Purpose?1) To prepare the structure of cells with minimum alteration from the

living state

2) To protect them against disruption during embedding and sectioning

3) To prepare specimen (usually tissue) for subsequent treatments including staining and exposure to the electron beam.

Satisfactory preservation and preparation of the cell, in order to see under the electron microscope.

Things need to know about fixing

• Fixing material or substances are called Fixative.• Ideal fixative – Kill tissues quickly?, cause minium

shrinkage or swelling.

-In order to kill quickly

1) Freeze drying (e.g our Embryo)

2) Chemical fixing (Sample 1,2,3)

- Speed of penetration

a) Low molecular weight e.g) Formaldehyde

Molecular weight – penetration speed? Not

always!! (e.g HgCl2)

Things need to know about fixing

b) Reacting Radicle

c) Solubility in lipids d) Polarity of molecule * Separation of liquid phase from solid phase of

protoplasm is an essential of fixation.

- Everything is in the water. We changing water to fixative solution to organic solvent to plastic. –Need stable bonds in order to prevent breaking chemical bonds, cell component’s translocation or extraction.

Fixatives should give strong chemical bonds between cell components.

Fixatives

1) Coagulant Fixatives – Flocculate all proteins e.g) Ethanol - Considerable change in protein structure. Most non additive.

2) Non-Coagulant Fixatives – Very little dissociation of protein from water. Protein retain at least some of their reactive group. e.g) glutraldehyde, OsO4, acrolein, formaldehyde. Most additive.

ProtocolFix: 2% paraformaldehyde + 0.1% glutraldehyde in 0.1M

cacodylate with 2mM CaCl2, pH 7.4 approx 1hr, r.t.Wash: 0.1M cacodylate with 2mM CaCl2 2*10minDehydrate: 10%,30,50,70,85,95,100% ethanol 5-10minInfiltrate: 1:1 ethanol/LR White 1-2hr on rotator 1:2 “ 100% LR White overnight on rotator 100%LR White 1-2 hr on rotatorEmbed: in gelatin capsules or in other molds where oxygen

can be excluded.Polymerize: with UV light in freezer or in oven at 50 C for

12-48 hours.

Factors affecting quality of fixation

1)pH – Normal cell’s average pH – 7.4 everythings are stable in this pH.

2)Buffer Type – types of ion presents affect fixation of specimen. Common buffers used for EM- Collidine, phosphate, Arsenate, Sodium bicarbonate, Veronal acetate, Chromatedichromate.(hand out)

3)Tonicity(Osmolarity) – Rate of penetration Iso?, Hypo?, Hyper? Achieved by addition of

electrolyte(e.g NaCl), Non electrolyte(e.g.

sucrose) Ca2+(prevent swell? And more?)

Factors affecting quality of fixation

4)Concentration of Fixative

5)Temperature and Duration of Fixation.

-Formaldehyde: penetrate rapidly but fix slowly

-Glutaraldegyde, Potassium permanganate: Penetrate slowly but fix rapidly.

Chemicals in Fixative

-Paraformaldehyde: penetrate fast, React with many functional groups on proteins including amine, thiol, hydroxyl,imidazol, phenolic group. Also can crosslink DNA. stabilize protein by crosslinking?

Chemicals in Fixative

- Glutaraldehyde: Cross link proteins rapidly and irreversibly. React with lysine, cysteine, histidine, tyrosine, tryptophan.

Chemicals in Fixative

- Osmium Tetraoxide (OsO4): Crosslink lipids, Reduced osmium can provide more electron density-can increase contrast. Most targets are unsaturated fatty

acid.

ProtocolFix: 2% paraformaldehyde + 0.1% glutraldehyde in 0.1M

cacodylate with 2mM CaCl2, pH 7.4 approx 1hr, r.t.Wash: 0.1M cacodylate with 2mM CaCl2 2*10minDehydrate: 10%,30,50,70,85,95,100% ethanol 5-10minInfiltrate: 1:1 ethanol/LR White 1-2hr on rotator 1:2 “ 100% LR White overnight on rotator 100%LR White 1-2 hr on rotatorEmbed: in gelatin capsules or in other molds where oxygen

can be excluded.Polymerize: with UV light in freezer or in oven at 50 C for

12-48 hours.

ProtocolFix: 2% paraformaldehyde + 0.1% glutraldehyde in 0.1M

cacodylate with 2mM CaCl2, pH 7.4 approx 1hr, r.t.Wash: 0.1M cacodylate with 2mM CaCl2 2*10minDehydrate: 10%,30,50,70,85,95,100% ethanol 5-10minInfiltrate: 1:1 ethanol/LR White 1-2hr on rotator 1:2 “ 100% LR White overnight on rotator 100%LR White 1-2 hr on rotatorEmbed: in gelatin capsules or in other molds where oxygen

can be excluded.Polymerize: with UV light in freezer or in oven at 50 C for

12-48 hours.

ProtocolFix: 2% paraformaldehyde + 0.1% glutraldehyde in 0.1M

cacodylate with 2mM CaCl2, pH 7.4 approx 1hr, r.t.Wash: 0.1M cacodylate with 2mM CaCl2 2*10minDehydrate: 10%,30,50,70,85,95,100% ethanol 5-10minInfiltrate: 1:1 ethanol/LR White 1-2hr on rotator 1:2 “ 100% LR White overnight on rotator 100%LR White 1-2 hr on rotatorEmbed: in gelatin capsules or in other molds where oxygen

can be excluded.Polymerize: with UV light in freezer or in oven at 50 C for

12-48 hours.

ProtocolFix: 2% paraformaldehyde + 0.1% glutraldehyde in 0.1M

cacodylate with 2mM CaCl2, pH 7.4 approx 1hr, r.t.Wash: 0.1M cacodylate with 2mM CaCl2 2*10minDehydrate: 10%,30,50,70,85,95,100% ethanol 5-10minInfiltrate: 1:1 ethanol/LR White 1-2hr on rotator 1:2 “ 100% LR White overnight on rotator 100%LR White 1-2 hr on rotatorEmbed: in gelatin capsules or in other molds where oxygen

can be excluded.Polymerize: with UV light in freezer or in oven at 50 C for

12-48 hours.

Sectioning• Cut with Ultra Microtome.

Sectioning

• Thick cut – 1micrometer(glass blade)

Sectioning

• Thin cut – 80nm

Immunolabeling Protocol1. Incubate on drops of 5% Na-metaperiodate 40ul 20min

2. Wash with dH2O 40ul 3*3

3. Block with PBST+5% milk 40ul 15min

4. Incubate with primary antibody 10ul 1hr(1:10)

5. Wash with PBST+5% milk 40ul 2*5min

6. Incubate with secondary antibody 10ul 30min(1:100)

7. Wash with PBST+5% milk 40ul 5min

8. Wash with PBS 50ul 2*5min

9. Wash with dH2O 50ul 2*10min

10. Poststain with UrAc and Pb citrate

11. Dry and view with TEM

Sodium Metaperiodate: Stablize polysaccharides and glycoprotein. Etching

Primary Ab: Ra anti GFP IgG

Secondary Ab: Goat anti Ra IgG conjugated with colloidal gold.

Colloidal gold

Gold particle, not have defined structure,found in suspension in water, negatively charged.

Liquid usually appear red color(particles less than 100nm), conjugated with monoclonal antibody’s Fc region by ionic interaction which lysine, tryptophan, and cysteine are found

Available by size.

We used 10nm.

Transmission Electron

Microscopy

Grid 2 Treated with tunicamycin

More grid2

Grid 4 Embryo

Poststain??

• UrAc and Pb citrate: heavy metal? Electron scattering power?

• Make a circle!! (penetration or not??)

• Unstained Stained

Discussion

• We didn’t even look at grid 1 and 3.

• We couldn’t find cluster of gold particle on our sample 2.

• We know GFP is in there by tracking with epi-fluorescence microscopy and western blot.

Conclusion:

Something wrong!!!

So, What can we do? Huh?

• It could be fixation problem.

• Maybe we washed too wrong.

• Maybe they are too shy.