Immunogold Electron Microscopy

Example: CHIFF labeling

1. The collected CHIFF was resuspended in TEBP at a density of 2 x 10⁹ cell equivalents (ceq)/ml. BSA and NaCl were added to 1mg/ml and 150mM, respectively.

2. Preabsorbed cAR1 antiserum was added at 1:200 dilution. Mixing was carried out for 1-2 hours at 4^oC.

3. After 3 washes in TEB plus NaCl and BSA, the sample was combined with 1:25 or 1:4 (for saturation staining) diluted gold-conjugated secondary antibody (10nm size, Amersham) in TEB plus NaCl and BSA and incubated at room temperature for 1-2 hours. Five washes were carried out in TEB.

Follow EM facility protocol, basically.

4. The final pellet was fixed with  fresh 2% glutaraldehyde in 50 mM cacodylate buffer  pH 6.8 (22^o for 30 minutes), postfixed with 1% OsO₄ (30 minutes on ice), and stained with EM facility stocks of uranyl acetate and lead citrate.

5. Wash 5x in dH₂0 and then dehydrate in at least one hour 50%, 70%, 90%, then 100% ethanol overnight.  Wash 3x at least one hour with propylene oxide and switch to PO.

6. Move to capsule and embed in Eponate (Pella).  At least one hour each 50% Ep:PO, 70% Ep:PO, 90% Ep:PO, then 100% Ep overnight. Bake overnight thin sections were cut.

7. Examined under a Philips 410 TEM. Control samples were prepared in parallel with preimmune serum to insure that the observed gold labelling was specifically dependent on the interaction of cAR1 with anti-cAR1 antibodies.

Example:  Whole cells

1.  Cells were plated onto 35 mm petri dishes and then fixed/permeabilized with 2% glutaraldehyde in 50 mM cacodylate buffer containing 1% CHAPS for 5 minutes.

Inclusion of CHAPS was necessary to permeabilize the cells in order for antibody molecules to access the C-terminus of cAR1.

2.  Cells were then postfixed in 0.5% glutaraldehyde for 10-15 minutes.

3.  The fixed cells were washed 2 x 15 minutes with 1 mg/ml NaBH₄ in water to quench glutaraldehyde, and then blocked for 30 minutes with 3% BSA in wash buffer.

4.  Affinity-purified cAR1 antibody (1:200 dilution) was added and incubated for 1 hour. 5) After three 5 minutes washes with wash buffer, 1:20 diluted secondary antibody-gold conjugate (5nm size, Amersham) was added and further incubated for 1 hour.

After six 5 minutes washes, cells were further fixed with 1% OsO₄.

Samples were then dehydrated, embedded in Eponate resin (Ted Pella), sectioned and post-stained with 1% uranyl acetate.

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This page was last edited 06/26/2003