Download Secret of TEM

Immunolabeling and Transmission
Electron Microscopy
Our Samples
1)
2)
3)
4)
GFP-Control (GFP – 2SC)
GFP-Treated with Tunicamycin
Wild Type
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
To protect them against disruption during embedding and
sectioning
To prepare specimen (usually tissue) for subsequent treatments
including staining and exposure to the electron beam.
2)
3)
 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.
Protocol
Fix: 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*10min
Dehydrate: 10%,30,50,70,85,95,100% ethanol 5-10min
Infiltrate: 1:1 ethanol/LR White 1-2hr on rotator
1:2
“
100% LR White overnight on rotator
100%LR White 1-2 hr on rotator
Embed: 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.
Protocol
Fix: 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*10min
Dehydrate: 10%,30,50,70,85,95,100% ethanol 5-10min
Infiltrate: 1:1 ethanol/LR White 1-2hr on rotator
1:2
“
100% LR White overnight on rotator
100%LR White 1-2 hr on rotator
Embed: 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.
Protocol
Fix: 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*10min
Dehydrate: 10%,30,50,70,85,95,100% ethanol 5-10min
Infiltrate: 1:1 ethanol/LR White 1-2hr on rotator
1:2
“
100% LR White overnight on rotator
100%LR White 1-2 hr on rotator
Embed: 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.
Protocol
Fix: 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*10min
Dehydrate: 10%,30,50,70,85,95,100% ethanol 5-10min
Infiltrate: 1:1 ethanol/LR White 1-2hr on rotator
1:2
“
100% LR White overnight on rotator
100%LR White 1-2 hr on rotator
Embed: 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.
Protocol
Fix: 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*10min
Dehydrate: 10%,30,50,70,85,95,100% ethanol 5-10min
Infiltrate: 1:1 ethanol/LR White 1-2hr on rotator
1:2
“
100% LR White overnight on rotator
100%LR White 1-2 hr on rotator
Embed: 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 Protocol
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Incubate on drops of 5% Na-metaperiodate 40ul 20min
Wash with dH2O 40ul 3*3
Block with PBST+5% milk 40ul 15min
Incubate with primary antibody 10ul 1hr(1:10)
Wash with PBST+5% milk 40ul 2*5min
Incubate with secondary antibody 10ul 30min(1:100)
Wash with PBST+5% milk 40ul 5min
Wash with PBS 50ul 2*5min
Wash with dH2O 50ul 2*10min
Poststain with UrAc and Pb citrate
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.