Novocastra Laboratories Ltd., Balliol Business Park West ...

Dysferlin
Lyophilized Mouse Monoclonal Antibody Product No: NCL-Hamlet-2
Intended Use
For in vitro diagnostic use.
NCL-Hamlet-2 is intended for the qualitative identification by light microscopy of the human dysferlin molecule in frozen and paraffin sections.
The clinical interpretation of any staining or its absence should be complemented by morphological studies using proper controls and should
be evaluated within the context of the patient’s clinical history and other diagnostic tests by a qualified pathologist.
Principle of Procedure
Immunohistochemical (IHC) staining techniques allow for the visualization of antigens via the sequential application of a specific antibody to
the antigen (primary antibody), a secondary antibody to the primary antibody with a chromogenic substrate with interposed washing steps.
The enzymatic activation of the chromogen results in a visible reaction product at the antigen site. The specimen may then be coverslipped.
Results are interpreted using a light microscope and aid in the differential diagnosis of pathophysiological processes, which may or may not
be associated with a particular antigen.
Clone
Ham3/17B2
Immunogen
Synthetic peptide containing amino acids 349-366, spanning exons 11 and 12, of the human dysferlin molecule.
Specificity
Reactive with the dysferlin molecule in human skeletal muscle. Also present in many non-muscle tissues.
Reagent Composition
NCL-Hamlet-2 is a lyophilized tissue culture supernatant containing 15mM sodium azide as a preservative. The user is required to
reconstitute the contents of the vial with the correct volume of sterile distilled water as indicated on the vial label.
Ig Class
IgG2b
Total Protein Concentration Total Protein
1.0 - 8.0 g/L. Refer to vial label for batch specific total protein concentration.
Antibody Concentration
Greater than or equal to 333.0 mg/L as determined by ELISA. Refer to vial label for batch specific Ig concentration.
Recommendations On Use
Immunohistochemistry (see D. Methodology) on frozen sections: Suggested dilution: 1:20 - 1:40 for 60 minutes at 25 o C. This is provided as a
guide and users should determine their own optimal working dilutions. NCL-Hamlet-2 requires the sections to be fixed in acetone/methanol at
a ratio of 1:1 for 4 minutes at 25 o C prior to incubation with the primary antibody.
Immunohistochemistry (see D. Methodology) on paraffin sections. Suggested dilution: 1:20 - 1:40 for 60 minutes at 25 o C. High temperature
antigen retrieval using 0.01M citrate retrieval solution (pH 6.0) is recommended. This is provided as a guide and users should determine their
own optimal working dilutions.
Storage and Stability
Store unopened antibody at 2-8 o C. Under these conditions, there is no significant loss in product performance up the expiry date indicated on
the vial label. Do not use after expiration date indicated on the container label. The reconstituted antibody is stable for at least two months
when stored at 2-8 o C. For long term storage, it is recommended that aliquots of the antibody are stored frozen at -20 o C (frost free freezers are
not recommended). Repeated freezing and thawing must be avoided. Prepare working dilutions on the day of use. Return to 2-8 o C
immediately after use. Storage conditions other than those specified above must be verified by the user.
Specimen Preparation
Freeze specimen tissue blocks in isopentane chilled in liquid nitrogen (see Warnings and Precautions) for frozen tissue sections. The
specimens do not require further fixation but should be embedded in OCT compound (Sakura, Product No. Tissue-Tek 4583).
The recommended fixative is 10% neutral-buffered formalin for paraffin-embedded tissue sections.
Warnings and Precautions
This reagent has been prepared from the supernatant of cell culture. As it is a biological product, reasonable care should be taken when
handling.
The molarity of sodium azide in this reagent is 15mM. A Material Safety Data Sheet (MSDS) is available upon request for sodium azide.
Consult federal, state or local regulations for disposal of any potentially toxic components.
Specimens, before and after fixation, and all materials exposed to them, should be handled as if capable of transmitting infection and
disposed of with proper precautions. 1 Never pipette reagents by mouth and avoid contacting the skin and mucous membranes with reagents
and specimens. If reagents or specimens come in contact with sensitive areas, wash with copious amounts of water. Seek medical advice.
Minimize microbial contamination of reagents or an increase in non-specific staining may occur.
Incubation times or temperatures, other than those specified, may give erroneous results. Any such changes must be validated by the user.
Liquid nitrogen due to its excessively cold temperature causes burns and protective clothing, including gloves and visor, should be used when
handling. Use in a well ventilated area.
Isopentane is highly flammable and harmful by ingestion and inhalation. It is also irritating to skin and eyes, and is a narcotic in high
concentration.
Quality Control
Differences in tissue handling and technical procedures in the user’s laboratory may produce significant variability in results, necessitating
regular performance of in-house controls in addition to the following procedures.
Controls should be fresh autopsy/biopsy/surgical specimens frozen as soon as possible in the same manner as the patient sample(s).
Positive Tissue Control
Used to indicate correctly prepared tissues and proper staining techniques.