Protocol created by Lorena Maestre - Monoclonal Antibodies Unit, Centro Nacional de Investigaciones Oncológicas
Preparation of the slides
- Frozen sections
- Cut 4-8 um thick cryostat sections and mount on superfrost plus slides.
- Air dry the slides overnight.
- Fix in acetone for 10 min and store at -20º or -80º until needed.
- Before staining, warm slides at room temperature.
- Paraffin sections
- Deparaffinize sections in xylene, 2x5min.
- Hydrate with 100% ethanol, 2x3min.
- Hydrate with 95% ethanol, 1min.
- Rinse in distilled water.
Pre-treatment of tissue sections
- Proceed to antigen retrieval pre-treatment as required (Only for paraffin-embedded sections).
- Serum Blocking: incubate sections with FCS for 30 min to block immunoglobulin non-specific binding.
- Primary Antibody: incubate sections with primary antibody at appropriate dilution in PBS + 10% FCS for 1 hour at room temperature.
- Wash slides in PBS + 0.5% Tween20, 3 times for 5 min each.
- Secondary Antibody: incubate sections for 1 hour with fluorochrome-conjugated antibodies against different immunoglobulin isotypes (dilution 1:200), diluted in PBS + 10% FCS. Keep the section in a humid chamber in the dark.
- Wash your slides in PBS + 0.5% Tween20, 3 times for 5 min each.
- Counterstain: add antifading and DAPI.
- Superfrost plus slides (Menzel-Glaser J1800AMNZ)
- Tween20 (Sigma P794-9)
- Fluorochrome-conjugated antibodies (Molecular Probes)
- Antifading (Qbiogene S36936)
- DAPI (Vector H-1200)