Fluorescent antibody techniques, direct and indirect, are standby procedures that remain unsurpassed for versatility and accurate detection of either antigen or antibodies. The FA technique offers rapid deployment of new assays with a minimum of development time. It has the distinct advantage over other assay methods of enabling the operator to visually distinguish between specific and non-specific reactions.
RECOMMENDED STAINING PROCEDURE FOR INDIRECT FA:
- Warm slide to room temperature before removing from foil pouch.
- Place 10-50 µl (depending on well size) diluted serum on the designated wells. Dilute serum in
FA Serum Diluting Buffer.
- Incubate slide in humid chamber at 37°C for 30 minutes.
- Using a wash bottle, gently rinse slide briefly in FA Rinse
Buffer, and then soak for 10 minutes in FA Rinse
Buffer.
- Drain slide and dry around wells by pressing blotter (included in the pouch) to front surface. Place 10-50 µl (depending on well size)
FITC-labeled anti-IgG or IgM conjugate on the wells.
- Incubate as in step 3.
- Rinse as in step 4.
- Drain slide and dry back and edges with a paper towel. Do not allow stained surface to dry. Do not rinse with water.
- Place a drop of FA Mounting Fluid per well, top with cover slip, and view with a good quality fluorescence microscope at 100X-250X. Confirmation may be made at 400X.
RECOMMENDED STAINING PROCEDURE FOR DIRECT FA:
- Warm slide to room temperature before removing from foil pouch.
- Place 10-50 µl (depending on well size) of direct FA conjugate on the designated wells.
- Incubate slide in humid chamber at 37°C for 30 minutes.
- Using a wash bottle, gently rinse slide briefly in FA Rinse
Buffer and then soak for 10 minutes in FA Rinse
Buffer.
- Drain slide and dry back and edges with a paper towel. Do not allow stained surface to dry. Do not rinse with water.
- Place a drop of FA Mounting Fluid
per well, top with cover slip, and view with a good quality fluorescence microscope at 100X-250X. Confirmation may be made at 400X.
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