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Recommendations On Use
Immunohistochemistry on parafn sections.
Heat Induced Epitope Retrieval (HIER): Please follow the instructions for use in Novocastra Epitope Retrieval Solution pH 9.
Suggested dilution:
1:2000 for 30 minutes at 25 °C. This is provided as a guide and users should determine their own optimal working
dilutions.
Visualization:
Please follow the instructions for use in the Novolink
™
Polymer Detection Systems. For further product information
or support, contact your local distributor or regional ofce of Leica Biosystems, or alternatively, visit the Leica Biosystems Web site,
www.LeicaBiosystems.com
The performance of this antibody should be validated when utilized with other manual staining systems or automated platforms.
Materials Provided
See Reagent.
Materials Required But Not Provided
See Novolink
™
Polymer Detection Systems Instructions for Use.
Quality Control
Differences in tissue processing and technical procedures in the user’s laboratory may produce signicant variability in results,
necessitating regular performance of in-house controls in addition to the following procedures.
Controls should be fresh autopsy/biopsy/surgical specimens, formalin-xed, processed and parafn wax-embedded 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.
One positive tissue control should be included for each set of test conditions in each staining run.
A tissue with weak positive staining is more suitable than a tissue with strong positive staining for optimal quality control and to detect
minor levels of reagent degradation.
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Recommended positive control tissue is lung (pneumocytes).
If the positive tissue control fails to demonstrate positive staining, results with the test specimens should be considered invalid.
Negative Tissue Control
Should be examined after the positive tissue control to verify the specicity of the labeling of the target antigen by the primary antibody.
Recommended negative control tissue is skin.
Alternatively, the variety of different cell types present in most tissue sections frequently offers negative control sites, but this should be
veried by the user.
Non-specic staining, if present, usually has a diffuse appearance. Sporadic staining of connective tissue may also be observed in
sections from excessively formalin-xed tissues. Use intact cells for interpretation of staining results. Necrotic or degenerated cells often
stain non-specically.
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False-positive results may be seen due to non-immunological binding of proteins or substrate reaction products.
They may also be caused by endogenous enzymes such as pseudoperoxidase (erythrocytes), endogenous peroxidase
(cytochrome C), or endogenous biotin (eg. liver, breast, brain, kidney) depending on the type of immunostain used. To differentiate
endogenous enzyme activity or non-specic binding of enzymes from specic immunoreactivity, additional patient tissues may be stained
exclusively with substrate chromogen or enzyme complexes (avidin-biotin, streptavidin, labeled polymer) and substrate-chromogen,
respectively. If specic staining occurs in the negative tissue control, results with the patient specimens should be considered invalid.
Negative Reagent Control
Use a non-specic negative reagent control in place of the primary antibody with a section of each patient specimen to evaluate
non-specic staining and allow better interpretation of specic staining at the antigen site.
Patient Tissue
Examine patient specimens stained with NCL-L-SP-A last. Positive staining intensity should be assessed within the context of any
non-specic background staining of the negative reagent control. As with any immunohistochemical test, a negative result means that
the antigen was not detected, not that the antigen was absent in the cells/tissue assayed. If necessary, use a panel of antibodies to
identify false-negative reactions.
Results Expected
Normal Tissues
Clone 32E12 detected surfactant protein A in the cytoplasm of pneumocytes within lung. (Total number of normal cases evaluated = 53).
Abnormal Tissues
Clone 32E12 stained 8/44 lung tumors (including 8/25 adenocarcinomas, 0/11 squamous cell carcinomas, 0/2 bronchiolalveolar
carcinomas, 0/2 large cell carcinomas, 0/2 large cell necroendocrine carcinomas and 0/2 small cell carcinomas).
No staining was detected in brain tumors (0/2), esophageal tumors (0/2), a laryngeal tumor (0/1), a thymic tumor (0/1), thyroid tumors
(0/3), breast tumors (0/2), gastric tumors (0/2), soft tissue tumors (0/2), tongue tumors (0/2) metastatic tumors of unknown origin (0/2),
liver tumors (0/4), renal tumors (0/2) ovarian tumors (0/4), cervical tumors (0/2), testicular tumors (0/2), colonic tumors (0/2), rectal
tumors (0/2) and skin tumors (0/2). (Total number of tumor cases evaluated = 83).