How to Use ONCYTE® Film-Wells / Film-Slides
A combination of our SecureSeal™ chambers and ONCYTE® nitrocellulose film coated slides that provides an easily removable, low volume chamber and the protein binding benefits of a nitrocellulose coating.
User Protocol: Instructions for Immunohistochemical Staining
- Apply specimen to Film area (see side A).
- Air dry
- Fix specimens using appropriate fixative for the antigen studied. Commonly recommended fixatives are:
A. Paraformaldehyde: 4% in phosphate buffered saline (PBS). 0.05M magnesium chloride, 0.3% Tween-20, pH 7.4. Fix at 4 C. Fixation time may vary according to specimen thickness and specific antigen.
B. Zamboni’s: Nature 216:174-175, 1967.
- Wash with three changes of PBS containing Tween-20 (0.1%) pH 7.5 (TPBS).
- Block non-specific binding of primary antibody: - peroxidase based detection: incubate specimens for 15 minutes with 0.3% hydrogen peroxide prepared in de-ionized water.
- alkaline phosphatase based detection: incubate specimens 15 minutes at room temperature in TPBS containing 1% normal serum from the species in which the secondary antibody is made.
- Blot excess serum from specimens.
- Incubate specimens for 30 minutes with primary antibody diluted in TPBS containing 1% serum.
- Wash Film-slides with 3 changes of TPBS.
- Incubate with secondary antibody diluted in TPBS containing 1% normal serum.
- Repeat Step 8.
Note: remaining steps refer to alkaline phosphatase based detection.
- Incubate specimens in alkaline phosphatase labeled reagent.
- Repeat Step 8.
- Equilibrate specimens for 2 minutes in 0.1M TRIS pH 9.5.
- Incubate in substrate or solution 20-30 minutes.
- Rinse with tap water.
- Counterstain if desired (see side A).
- Air dry or dehydrate through isopropyl alcohol series.
- Clear with 3 changes of xylenes.
- Coverslip using non-aqueous mounting medium.
How to Use ONCYTE® Film-Wells / Film-Slides
- Specimens may be applied to a Film-Slide by any conventional method including centrifugation, cryostat mounting, tissue printing pipetting, and array depositors.
- Film-Slides provide superior specimen adherence, out performing silane and protein treated glass slides.
- Soluble analyte ay be bound to Film-slides by air drying, UV crosslinking, baking or microwaving. Film-slides are autoclavable.
- Specimens may be chemically fixed using aqueous fixatives or compatible alcohols.
Film-slides are ideally suited for use in most cytochemistry assay protocols including ICC, ISH, ISPCR and HTS.
- Film-slides are chemically resistant to reagents typically used in most assays for cell analysis including 50% formamide.
- Film-slides are not recommended for use with acetone, ethanol & methanol. Isopropanol and butanol are common substitutes.
- Bi-directional access to specimen by reagents may reduce incubation times and reagent concentrations.
- Film-slides are compatible with a variety of counterstains includng Mayer’s hematoxylin, nuclear fast red and light green SF. Gill’s hematoxylin is recomended (0.05% HCl may be used to de-stain). Eoin (0.5%, diluted in isopropanol). In some cases counterstains may bind to the film, this usually does not interfere with the microscopic examination of specimens.
- Film-Slides are made transparent for microscopic and imaging applications by xylenes or immersion oil with a refractive index of 1.515. For aqueous mounting Vectashield (Vector Labs), Slow Fade & Slow Fade Light (Molecular Probes, Inc.) are recommended.
- Permanent sections may be made by coverslip mounting specimens using non-aqueous media after dehydration through alcohol or by air drying.
References:
High-Definition Cell Analysis In Situ Using Microporous Films. Cel Vision 2:499-509, 1995.
Cytometrically Coherent Transfer of Receptor Proteins on Microporous Membranes. BioTechniques 20:641-650. 1991.
ONCYTE® Film-Slides & Film-Wells are Intended for Laboratory Use Only
ONCYTE® is a registred trademark of Grace Bio-Labs, Inc.
Online Ordering
SecureSeal™ chambers and ONCYTE® are available online from the EMS Catalog. For ordering or product information, click SecureSeal™ or ONCYTE® .