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Johns Hopkins University School of Medicine
Department of Cell Biology
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Baltimore, MD 21205
 

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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 4oC.

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 (22o 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