Fix the cell preparation (cell culture or tissue sections) for 15 minutes at room temperature using one of
the fi xatives listed below:
- 2% paraformaldehyde in PBS
- 9:1 (v/v) acetone/water solution
- Pure isopropanol solution
- Absolute ethanol solution
Then rinse with PBS.
Dilute the reagent twentyfold in a PBS-Evans Blue solution prepared according to the following formula:
PBS-Evans Blue
________________
NaCl: 8 gm
KH2PO4: 0.2 gm
KCl: 0.2 gm
Na2HPO4 . 2H2O: 1.15 gm
Evans Blue: 0.01 gm
NaN3: 0.1 gm
H20 1 L
Incubate the preparation on the sample for 1 hour at room temperature, preferably in a humidity chamber.
Upon completion of this incubation period rinse the preparation with a PBS solution.
Then add the conjugate (fl uorescein-labelled anti-mouse immunoglobulin) at the manufacturer’s recommended
dilution. The conjugate available from Bio-X Diagnostics (Bio 305) should be diluted twentyfold
in PBS-Evans Blue solution.
Incubate the preparation on the sample for 1 hour at room temperature, preferably, in a humidity
chamber.
After this second incubation step rinse the preparation with PBS.
Dry the slide, then add the mounting medium made up as follows:
anti-Mouse Ig FITC or
peroxidase
anti-CSFV
Tissue section
Bio-X Diagnostics
Site du complexe des Postes 49, rue Joseph Wauters BE-5580 Jemelle BELGIUM (Europe)
Tel: +32 (0)84.32.23.77 Fax: +32 (0)84.31.52.63 E-mail: info@biox.com http://www.biox.com
www.biox.com
Mounting medium
_________________
Glycerol 9 parts by volume
PBS 1 part by volume
Place a cover slip on the slide, then observe under a microscope fi tted for fl uorescence detection.
The antibody may be kept in its original vial at 4°C for more than a year. Never freeze this reagent.
Once diluted in the PBS-Evans Blue solution, the antibody remains stable for one week at 4°C.
II – INDIRECT IMMUNOPEROXIDASE ASSAY PROCEDURE
Fix the cell preparation (cell culture or tissue sections) for 15 minutes at room temperature using one of
the following fi xatives:
- 2% paraformaldehyde in PBS
- 9:1 (v/v) acetone/water solution
- Pure isopropanol solution
- Absolute ethanol solution
Then rinse with PBS.
Dilute the reagent twentyfold in PBS prepared according to the following formula:
PBS
________________
NaCl: 8 gm
KH2PO4: 0.2 gm
KCl: 0.2 gm
Na2HPO4 . 2H2O: 1.15 gm
NaN3: 0.1 gm
H20 1 L
Incubate the preparation on the sample for 1 hour at room temperature, preferably in a humidity chamber.
Upon completion of this incubation period rinse the preparation with PBS.
Then add the conjugate (peroxidase-coupled anti-mouse immunoglobulin) at the manufacturer’s recommended
dilution. The conjugate available from Bio-X Diagnostics (Bio 269) should be diluted fi ftyfold in
PBS.
Incubate the preparation on the sample for 1 hour at room temperature, preferably in a humidity chamber.
After this second incubation step rinse the preparation with PBS.
Then add the chromogen (AEC, precipitating TMB, DAB, etc.) and the substrate (hydrogen peroxide)
according to the manufacturer’s instructions. Examine under the microscope for the presence of the
coloured marker.
COMPOSITION: One vial of 500 μl
STORING THE REAGENT: The antibody must be stored at 4°C. It must never be frozen.
STABILITY: One year at 4°C
