Protocols
Immunoflourescence | Immunogold |
Silver Proteinate Charbohydate | DNA Specific Stains
Immunofluorescence Protocol
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Fix cell in 1-4% formaldehyde in growth medium or Phosphate Buffer (PBS w/o salt) or PHEM buffer for 20 min.Wash cells in growth medium, Phosphate Buffer or PHEM, 2X with gentle centrifugation or suction.
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Place cells on poly-l-lysine coated slide (1mg/ml poly-lysine) for a period of time. Place a drop of fixative (same as above) on cells.
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Wash cells in growth medium, Phosphate Buffer or PHEM, 2X with gentle centrifugation or suction.
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Wash Cells with PBS or with PHEM-DMOS, 2-3X for 5 minutes each wash.
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Place a drop of cells onto poly-l-lysine slides (if not already on). Let cells sit for 1/2hr, then remove excess fluid and allow to air dry (~5min).
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Add 1/50 to 1/100 dilution of primary anti-body in PBS or PHEM-DMSO with 0.1% BSA to cells on slide. Incubate at 37 o C for 30minutes.
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Suction off primary antibody and wash slide in PBS (or PHEM-DMSO) 3X for 5 minutes each.
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Add 1/50 to 1/100 dilution of secondary antibody in PBS (or PHEM-DMSO). Incubate at 37 o C for 30 minutes.
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Suction off secondary antibody and wash slide with PBS (or PHEM-DMSO), 3X for 5 min.
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Place mountant fluid on each well with cells, place coverslip over cells. Allow for diffusion of mountant for 1 minute and seal with nail polish.
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Store in refrigerator
Immunogold Protocolfor TEM
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Incubate grids in 50mM NH4Cl for up to 30 minutes (anything fixed with aldehydes) or in 5% H2O2 (use with osmicated samples)
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Wash grids in water and blot dry.
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Incubate grids in 5% low/non fat Carnation Instant Milk in PBST buffer for 30 min.
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Wash grids and blot dry.
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Incubate grids on 1 O antibody dilutes 1/50 (or whatever is recommended) in PBST for 1-4 hours in 37C or overnight at 4C.
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Wash grids in PBST for 30s and blot dry.
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Incubate grids in 2 O antibody in PBST (1/50 or 1/100) for 30-60minutes at 37C.
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Wash in PBST 3X for 30s
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Wash in DH2O 3X for 30s
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Blot dry and stain in UA/ Pb Citrate
Immunogold Solutions
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PBS Stock: 10nM Na2PO4, 500mM NaCl, 0.1% Tween 20
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PBS: 25ml of PBS Stock and add 225ml of dH20
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PBST: add 0.1ml of Tween 20 to 100ml of PBS
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Carnation Milk Block: add 2.5g or Carnation Instant Milk to 50 ml of PBST
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1' antibody solution
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1/50- 10ul of antibody to 0.5ml PBST
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1/100-10ul of antibody to 1ml PBST
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- 2' antibody solution 1/50- 10ul of anti-rat or anti-mouse antibody to 0.5ml of PBST(AB labeled with 10-20nm gold particles)
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NH4CL(50mM): 0.135g of NH4CL in 50ml of DH2O
Silver Proteinate Staining-carbohydrate Staining for TEM
Staining indicates the presence of polysaccharides, glycoprotein or glycolipids containing vicinal hydroxyl groups
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Collect sections on gold or nickel grids.
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Float grids on drops of 1% Periodic Acid* for 30min at RT
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Wash grids on drops of dH2O (3 x 10min)
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Float grids on 0.2% TCH in 20% acetic acid** for 30min to 24hrs.
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Wash grids in decreasing concentrations of acetic acid 20% -> 15% -> 10% -> 5% for 10 minutes each step. Then wash in dH2O (2 x15min)
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Stain with 1% silver proteinate*** for 30min at RT in a dark, humid chamber.
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Wash grids with dH2oO (2 x15min). Dry and observe on TEM. A fine electron deposit indicates positive staining.
*1% periodic acid is stable form 1 month at 4C. Corrosive
**0.2% TCH in 20% acetic acid is stable for several days at 4C
***1% silver proteinate should be prepared in advance (~12hr). Sprinkle powder into water and leave to stand for 30minutes. Centrifuge prior to use. Light sensitive. Can be stored for 1 week at 4C
DNA Specific Stains
SYTO-9* & Propidium Iodide**
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Dilute 1 m l of stock in 1ml of growth media with cells
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Stain for 2-5 minutes.
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Wash 3X with media
*Stains live cells, use blue excite
**Use green excite