ImmunoGold (Silver Staining) Incubation Protocol for General Applications
The procedure is based on a two-step incubation using a primary antibody and a gold-labeled secondary antibody conjugate. Steps 9 and 10 apply only when using Ultra Small-Grade reagents with Silver Enhancement and are skipped when using EM-Grade reagents.
Incubation Buffer System:
PBS, (10 mM Phosphate buffer, 150 mM NaCl), pH 7.4
0.1-0.2% AURION BSA-c
15 mM NaN3
Check the pH and adjust to 7.4 if necessary
Buffers are either prepared immediately before use or thawed from aliquots stored at -20°C.
Procedure for Single Labeling
Low Molecular Weight Block Step
To inactivate residual aldehyde groups present after aldehyde fixation incubate for EM: with 0.05 M glycine or lysine in PBS buffer for 15 minutes for LM: with 0.1% NaBH4 in PBS for 15 minutes
High Molecular Weight Block Step
PBS buffer with 5% BSA and 0.1% CWFS gelatin supplemented with 5% normal serum, for 30 minutes (same species as the antibody in the second immuno incubation step).
Incubation buffer for 5 minutes
Primary antibody incubation (see also *)
Incubate with a dilution of specific primary antibody, preferably affinity-purified, 1-5 µg/ml in incubation buffer, (or a high dilution of a high titre antiserum), for 30 minutes to 1 hour.
Antibody concentration and incubation time may have to be adapted according to the specific characteristics of the primary antibody and the specimen. If longer incubation times are required (e.g. with low titre antisera) the procedure should be carried out at 4°C overnight.
Incubation buffer for EM: 3 x 5 minutes for LM: 4 x 10 minutes
ImmunoGold conjugate incubation
Incubate with the gold conjugate reagent, dilution 1/20-1/40 (EM-grade reagents) or dilution 1/50-1/100 (Ultra Small reagents) in incubation buffer for 2 hours.
Incubation buffer for EM: 6 x 5 minutes for LM: 4 x 10 minutes
Further wash steps
PBS, 3x5 minutes, postfix in 2% glutaraldehyde in PBS, 5 minutes, PBS for 5 minutes and finally distilled water for 5x2 minutes.
Silver Enhancement, using
AURION R-GENT for Light Microscopy or BioAssays, or Aurion R-Gent SE-EM Silver enhancement procedure for Electron Microscopy Ref.: Danscher, G. Histochemistry 71, 1981, page 81-88.
Distilled water for 5x2 minutes.
Contrasting or Staining
*Remark: when performing pre-embedding labeling the time involved with incubation and washing may have to be prolonged to warrant complete penetration of reagents to internal antigens and removal of unreacted reagents!
For double labeling using secondary antibody immunogold EM-grade conjugates, two primary antibodies produced in different animal species are mixed and applied simultaneously (step 4). After the washing step (5), a mixture of the corresponding immunogold EM-grade reagents with two non-overlapping sizes is applied (step 6).