Protocols

Immunoflourescence | Immunogold |

Silver Proteinate Charbohydate | DNA Specific Stains

Immunofluorescence Protocol

1. 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.

   1. 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.

2. Wash cells in growth medium, Phosphate Buffer or PHEM, 2X with gentle centrifugation or suction.

3. Wash Cells with PBS or with PHEM-DMOS, 2-3X for 5 minutes each wash.

4. 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).

5. 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.

6. Suction off primary antibody and wash slide in PBS (or PHEM-DMSO) 3X for 5 minutes each.

7. Add 1/50 to 1/100 dilution of secondary antibody in PBS (or PHEM-DMSO). Incubate at 37 o C for 30 minutes.

8. Suction off secondary antibody and wash slide with PBS (or PHEM-DMSO), 3X for 5 min.

9. 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.

10. Store in refrigerator

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Immunogold Protocolfor TEM

1. Incubate grids in 50mM NH4Cl for up to 30 minutes (anything fixed with aldehydes) or in 5% H2O2 (use with osmicated samples)

2. Wash grids in water and blot dry.

3. Incubate grids in 5% low/non fat Carnation Instant Milk in PBST buffer for 30 min.

4. Wash grids and blot dry.

5. 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.

6. Wash grids in PBST for 30s and blot dry.

7. Incubate grids in 2 O antibody in PBST (1/50 or 1/100) for 30-60minutes at 37C.

8. Wash in PBST 3X for 30s

9. Wash in DH2O 3X for 30s

10. Blot dry and stain in UA/ Pb Citrate

Immunogold Solutions

• PBS Stock: 10nM Na2PO4, 500mM NaCl, 0.1% Tween 20

• PBS: 25ml of   PBS Stock and add 225ml of dH20

• PBST: add 0.1ml of Tween 20 to 100ml of PBS

• Carnation Milk Block: add 2.5g or Carnation Instant Milk to 50 ml of PBST

• 1' antibody solution

  • 1/50- 10ul of antibody to 0.5ml PBST

  • 1/100-10ul of antibody   to 1ml PBST

• 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)

• NH4CL(50mM): 0.135g of NH4CL in 50ml of DH2O

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Silver Proteinate Staining-carbohydrate Staining for TEM

Staining indicates the presence of polysaccharides, glycoprotein or glycolipids containing vicinal hydroxyl groups

1. Collect sections on gold or nickel grids.

2. Float grids on drops of 1% Periodic Acid* for 30min at RT

3. Wash grids on drops of dH2O (3 x 10min)

4. Float grids on 0.2% TCH in 20% acetic acid** for 30min to 24hrs.

5. Wash grids in decreasing concentrations of acetic acid 20% -> 15% -> 10% -> 5% for 10 minutes each step. Then wash in dH2O (2 x15min)

6. Stain with 1% silver proteinate*** for 30min at RT in a dark, humid chamber.

7. 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 

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DNA Specific Stains

SYTO-9* & Propidium Iodide**

1. Dilute 1 m l of stock in 1ml of growth media with cells

2. Stain for 2-5 minutes.

3. Wash 3X with media

*Stains live cells, use blue excite

**Use green excite

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