CMC35229020 • EN Rev. 2.0 • p.

Napsin A (MRQ-60)
Mouse Monoclonal Antibody
For In Vitro Diagnostic Use (IVD)

DIL concentrate dilution range

Intended Use
This antibody is intended for in vitro diagnostic (IVD) use.
Napsin A (MRQ-60) Mouse Monoclonal Primary Antibody is intended for laboratory use in
the detection of the Napsin A protein in formalin-fixed, paraffin-embedded tissue stained
in qualitative immunohistochemistry (IHC) testing. The results using this product should be
interpreted by a qualified pathologist in conjunction with the patient’s relevant clinical history,
other diagnostic tests and proper controls.

Summary and Explanation

Napsin is a pepsin-like aspartic proteinase in the A1 clan of the AA clade of proteinases.1-3
There are two closely related napsins, napsin A (NAPSA) and napsin B (NAPSB).1-3 Napsin A is
involved in processing propeptide pulmonary surfactant protein B (proSP-B) in the lung.4 In
normal tissue, Napsin A is expressed in type II pneumocytes of the lung and proximal tubules
of the kidney.1-3 Napsin A is a useful marker for lung adenocarcinoma.1-3, 5-8

Principles and Procedures

The stated primary antibody may be used as the primary antibody for immunohistochemical
staining of formalin-fixed, paraffin-embedded tissue sections. In general, immuno­histo­
Product Identification chemical staining in conjunction with a HRP or Alk Phos linked detection system allows
the visualization of antigens via the sequential application of a specific antibody (primary
Description antibody) to the antigen, a secondary antibody (link antibody) to the primary antibody, an
enzyme complex and a chromogenic substrate with interposed washing steps. The enzymatic
352M-94 0.1 ml concentrate activation of the chromogen results in a visible reaction product at the antigen site. The
352M-95 0.5 ml concentrate specimen may then be counterstained and a coverslip applied. Results are interpreted using a
352M-96 1.0 ml concentrate light microscope.
352M-97 1.0 ml predilute ready-to-use
352M-98 7.0 ml predilute ready-to-use
Materials and Methods
352S Positive control slides, 5 slides/pack
352M-90 25.0 ml predilute ready-to-use Reagents Provided

Product Composition
Symbol Definitions
Predilute: diluted in Tris Buffer, pH 7.3-7.7, with 1% BSA and
<0.1% Sodium Azide
keycode
Concentrate: diluted in Tris Buffer, pH 7.3-7.7, with 1% BSA and
P predilute <0.1% Sodium Azide
C concentrate Host Mouse
A ascites Isotype IgG1/k
E serum Recommended working dilution range 1:100-1:500
S supernatant
 CMC35229020 • EN Rev. 2.0 • p. 2

Every antibody reagent is expiration dated. When properly stored, the reagent is stable to the
Product Composition (continued)
date indicated on the label. Do not use reagent beyond the expiration date for the prescribed
Source Supernatant storage method.

See product label for lot specific information for the following: There are no definitive signs to indicate instability of this product; therefore, positive and
negative controls should be run simultaneously with unknown specimens. Contact Cell
1. Antibody immunoglobulin concentration
Marque customer service if there is a suspected indication of reagent instability.
2. Source details

Specimen Collection and Preparation for Analysis

Reconstitution, Mixing, Dilution, Titration Routinely processed, neutral-buffered formalin-fixed, paraffin-embedded, tissues are suitable
Prediluted antibody is ready-to-use and optimized for staining. No reconstitution, mixing, for use with this primary antibody when used with Cell Marque detection kits (see Materials,
dilution, or titration is required. The concentrated antibody is optimized to be diluted to within Reagents, and Equipment Needed But Not Provided section). Note: Cell Marque evaluates
the dilution range using Cell Marque Diamond Diluent. performance only on human tissues. The recommended tissue fixative is 10% neutral-
buffered formalin. Variable results may occur as a result of prolonged fixation or special
Materials and Reagents Needed But Not Provided processes such as decalcification of bone marrow preparations.

The following reagents and materials may be required for staining but are not provided with Each section should be cut to the appropriate thickness (approximately 3 µm) and placed on a
the primary antibody: positively charged glass slide. Slides containing the tissue section may be baked for at least 2
hours (but not longer than 24 hours) in a 53-65°C oven.
1. Positive and negative control tissue
2. Microscope slides, positively charged
3. Drying oven capable of maintaining a temperature of 53-65°C Warnings and Precautions
4. Staining jars or baths 1. Take reasonable precautions when handling reagents. Use disposable gloves and lab coats
5. Timer when handling suspected carcinogens or toxic materials (example: xylene).
6. Xylene or xylene substitute 2. Avoid contact of reagents with eyes and mucous membranes. If reagents come in contact
7. Ethanol or reagent alcohol with sensitive areas, wash with copious amounts of water.
Note: Cell Marque’s one-step pretreatment, Trilogy™ (cat. #920P-06), can replace both 6 and 7 3. Patient specimens and all materials contacting them should be handled as biohazardous
above. materials and disposed of with proper precautions. Never pipette by mouth.
8. Deionized or distilled water
4. Avoid microbial contamination of reagents, as this could produce incorrect results.
9. Heating Equipment, such as Electric Pressure Cooker (cat. #976L), for tissue pretreatment
step 5. The user must validate incubation times and temperatures.
10. Detection system, such as HiDef Detection™ HRP Polymer System (cat. #954D-20) or 6. The prediluted, ready-to-use reagents are optimally diluted, and further dilution may
HiDef Detection™ Alk Phos Polymer System (cat. #962D-20) result in loss of antigen staining.
11. Chromogen, such as DAB Substrate Kit (cat. #957D-20) or Permanent Red Chromogen Kit 7. The concentrated reagents may be diluted optimally based on validation by user. Any
(cat. #960D-10) diluent used that is not specifically recommended herein must likewise be validated by
12. TBS IHC Wash Buffer + Tween®* 20 (cat. #935B-09) the user for both its compatibility and effect on stability.
13. Hematoxylin (cat. #930B-05) or other counterstain
8. When used according to instructions, this product is not classified as a hazardous
14. Antibody diluents, such as Diamond: Antibody Diluent (cat. #938B-05) or substance. The preservative in the reagent is less than 0.1% sodium azide and does not
Emerald: Antibody Diluent (cat. #936B-08) meet the OSHA (USA) criteria for hazardous substance at the stated concentration. See
15. Peroxide Block (cat. #925B-05) for use with HRP SDS.
16. Avidin-Biotin Blocking Reagents (cat. # 928B-02 for use with streptavidin-biotin 9. The user must validate any storage conditions other than those specified in the package
detection) insert.
17. Negative Control Reagent (cat. #932B-02 for mouse; cat. #933B-02 for rabbit,
cat. #939B-02 for universal) 10. Diluent may contain bovine serum albumin and supernatant may contain bovine serum.
The products containing fetal bovine serum and products containing bovine serum
18. Permanent Aqueous Mounting Medium (cat. #931B-03)
albumin are purchased from commercial suppliers. Certificates of Origin for the animal
19. Cover glass source used in these products are on file at Cell Marque. The certificates support that the
20. Light microscope (40-400x) bovine sources are from countries with negligible BSE risk and state sources of bovine from
USA and Canada.
Storage and Handling 11. As with any product derived from biological sources, proper handling procedures should
Store at 2-8°C. Do not freeze. be used.
To ensure proper reagent delivery and stability of the antibody after every run, the cap must
be replaced and the bottle must be immediately placed in the refrigerator in an upright
position.

Napsin A (MRQ-60) Antibody

 CMC35229020 • EN Rev. 2.0 • p. 3

Instructions For Use Negative Tissue Control

Recommended Staining Protocols for the stated primary antibody: The same tissue used for the positive tissue control may be used as the negative tissue control.
The variety of cell types present in most tissue sections offers internal negative control sites,
but this should be verified by the user. The components that do not stain should demonstrate
HiDef Detection™ HRP: the absence of specific staining, and provide an indication of non-specific background
HiDef Detection™ HRP Polymer System (cat. #954D-20) staining. If specific staining occurs in the negative tissue control sites, results with the patient
specimens must be considered invalid.
1 Epitope Retrieval Technique: HIER, Epitope Retrieval Reagent: Trilogy
2 Antibody Incubation Time (Minutes): 10-30
3 HiDef Detection Amplifier Incubation Time (Minutes): 10 Unexplained Discrepancies
4 HiDef Detection Polymer Detector Incubation Time (Minutes): 10 Unexplained discrepancies in controls should be referred to Cell Marque Customer Service
5 DAB Incubation Time (Minutes): 1-10 immediately. If quality control results do not meet specifications, patient results are invalid.
See the Troubleshooting section of this insert. Identify and correct the problem, then repeat
6 Dehydrate and Coverslip. the entire procedure with the patient samples.

HiDef Detection™ Alk Phos: Negative Control Reagent

HiDef Detection™ Alk Phos Polymer System (cat. #962D-20) A negative control reagent must be run for every specimen to aid in the interpretation of
results. A negative control reagent is used in place of the primary antibody to evaluate
1 Epitope Retrieval Technique: HIER, Epitope Retrieval Reagent: Trilogy nonspecific staining. The slide should be treated with negative control reagent, matching
2 Antibody Incubation Time (Minutes): 10-30 the host species of the primary antibody, and ideally having the same IgG concentration.
3 HiDef Detection Amplifier Incubation Time (Minutes): 10 The incubation period for the negative control reagent should equal the primary antibody
4 HiDef Detection Polymer Detector Incubation Time (Minutes): 10 incubation period.
5 DAB Incubation Time (Minutes): 15-30
6 Dehydrate and Coverslip. Interpretation of Results
The immunostaining procedure causes a colored reaction product to precipitate at the
Quality Control Procedures antigen sites localized by the primary antibody. Refer to the appropriate detection system
package insert for expected color reactions. A qualified pathologist experienced in
Positive Tissue Control immunohistochemistry procedures must evaluate positive and negative tissue controls before
A positive tissue control must be run with every staining procedure performed. This tissue may interpreting results.
contain both positive and negative staining cells or tissue components and serve as both the
positive and negative control tissue. Control tissues should be fresh autopsy, biopsy or surgical Positive Tissue Control
specimens prepared or fixed as soon as possible in a manner identical to the test sections. Use
of a tissue section fixed or processed differently from the test specimen will serve to provide The stained positive tissue control should be examined first to ascertain that all reagents are
control for all reagents and method steps except fixation and tissue processing. functioning properly. The presence of an appropriately colored reaction product within the
target cells is indicative of positive reactivity. Refer to the package insert of the detection
A tissue with weak positive staining is more suitable for optimal quality control and for system used for expected color reactions. Depending on the incubation length and potency
detecting minor levels of reagent degradation. Positive tissue control for the stated primary of the hematoxylin used, counterstaining will result in a pale to dark blue coloration of cell
antibody may include the following: nuclei. Excessive or incomplete counterstaining may compromise proper interpretation of
results. If the positive tissue control fails to demonstrate appropriate positive staining, any
results with the test specimens are considered invalid.
Positive Tissue Control
Tissue Visualization
Negative Tissue Control
Lung Adenocarcinoma Cytoplasmic The negative tissue control should be examined after the positive tissue control to verify
Kidney Cytoplasmic the specific labeling of the target antigen by the primary antibody. The absence of specific
staining in the negative tissue control confirms the lack of antibody cross reactivity to cells or
Renal Cell Carcinoma Cytoplasmic cellular components. If specific staining occurs in the negative tissue control, results with the
patient specimen are considered invalid. Nonspecific staining, if present, will have a diffuse
Known positive tissue controls should be utilized only for monitoring the correct performance appearance. Sporadic light staining of connective tissue may also be observed in sections
of processed tissues and test reagents, not as an aid in determining a specific diagnosis from tissues that are not optimally fixed. Intact cells should be used for interpretation of
of patient samples. If the positive tissue controls fail to demonstrate appropriate positive staining results. Necrotic or degenerated cells show non-specific staining.
staining, results with the test specimens must be considered invalid.
Patient Tissue
Patient specimens should be examined last. Positive staining intensity should be assessed
within the context of any background staining of the negative reagent control. As with any

Napsin A (MRQ-60) Antibody

 CMC35229020 • EN Rev. 2.0 • p. 4

immunohistochemical test, a negative result means that the antigen in question was not 13. When used in blocking steps, normal sera from the same animal source as the secondary
detected, not that the antigen is absent in the cells or tissue assayed. A panel of antibodies antisera may cause false negative or false positive results because of the effect of
may aid in the identification of false negative reactions (see Summary of Expected Results autoantibodies or natural antibodies.
section). The morphology of each tissue sample should also be examined utilizing a 14. False positive results may be seen because of non immunological binding of proteins
hematoxylin and eosin stained section when interpreting any immunohistochemical result. or substrate reaction products. They may also be caused by pseudoperoxidase activity
The patient’s morphologic findings and pertinent clinical data must be interpreted by a (erythrocytes), endogenous peroxidase activity (cytochrome C), or endogenous biotin
qualified pathologist. (example: liver, brain, breast, kidney) subject to the type of immunostaining technique
used.
Limitations 15. As with any immunohistochemistry test, a negative result means that the antigen was not
1. Color does not affect performance. detected, not that the antigen was absent in the cells or tissue assayed.

2. This reagent is “for professional use only” as immunohistochemistry is a multiple step 16. The prediluted antibody products are optimized as a ready-to-use product. Because of
process that requires specialized training in the selection of the appropriate reagents, the possibility of variation in tissue fixation and processing, it may be necessary to increase
tissues, fixation, processing; preparation of the immunohistochemistry slide; and or decrease the primary antibody incubation time on individual specimens.
interpretation of the staining results. 17. The antibody, in combination with detection systems and accessories, detects antigen(s)
3. For laboratory use only. that survive routine formalin fixation, tissue processing and sectioning. Users who
deviate from recommended test procedures remain, as they would in any circumstance,
4. For in vitro diagnostic use. responsible for interpretation and validation of patient results.
5. Tissue staining is dependent on the handling and processing of the tissue prior to
staining. Improper fixation, freezing, thawing, washing, drying, heating, sectioning, or
contamination with other tissues or fluids may produce artifacts, antibody trapping,
Summary of Expected Results
or false negative results. Inconsistent results may result from variations in fixation and
embedding methods, as well as from inherent irregularities within the tissue. See the following tables of reactivity:
6. Excessive or incomplete counterstaining may compromise proper interpretation of results. Normal Study
7. The clinical interpretation of any positive staining, or its absence, must be evaluated within # Stained
the context of clinical history, morphology, other histopathological criteria as well as other Tissue (+) Total # Notes
diagnostic tests. This antibody is intended to be used in a panel of antibodies if applicable. Brain 0 1
It is the responsibility of a qualified pathologist to be familiar with the antibodies, Adrenal Cortex 0 1
reagents, diagnostic panels, and methods used to produce the stained preparation. Ovary 0 1
Staining must be performed in a certified, licensed laboratory under the supervision of a Pancreas 0 1
pathologist who is responsible for reviewing the stained slides and assuring the adequacy
Parathyroid 0 1
of positive and negative controls.
Pituitary 0 1
8. Cell Marque provides antibodies and reagents at optimal dilution for use as instructed. Any Testis 0 1
deviation from recommended test procedures may invalidate expected results. Appropriate Thyroid 0 1
controls must be employed and documented. Users in any circumstance must accept
Breast 0 2
responsibility for interpretation of patient results.
Spleen 0 1
9. Cell Marque provides primary antibodies in concentrated format so that the user may Tonsil 0 1
subsequently optimally dilute for use subject to the user’s determination of and adherence Thymus 0 1
to suitable validation techniques. Users must validate the use of any diluents other than Bone Marrow 0 1
what is recommended herein. Once the primary is validated to be suitable for use, any
Lung 2 2
deviation from recommended test procedures may invalidate expected results. Appropriate
Heart 0 1
controls must be employed and documented. Users in any circumstance must accept
responsibility for interpretation of patient results. Esophagus 0 1
Stomach 0 1
10. This product is not intended for use in flow cytometry. Small Intestine 0 1
11. Reagents may demonstrate unexpected reactions in previously untested tissues. The Colon 0 1
possibility of unexpected reactions even in tested tissue groups cannot be completely Liver 0 1
eliminated because of biological variability of antigen expression in neoplasms, or Salivary Gland 0 1
other pathological tissues. Contact Cell Marque customer service with any suspected, Gall Bladder 0 1
documented unexpected reactions.
Kidney 3 3
12. Tissues from persons infected with hepatitis B virus and containing hepatitis B surface Bladder 0 1
antigen (HBsAg) may exhibit nonspecific staining with horseradish peroxidase. Prostate 0 2
Uterus 0 2

Napsin A (MRQ-60) Antibody

 CMC35229020 • EN Rev. 2.0 • p. 5

Normal Study (continued) 4. If all of the paraffin has not been removed, the deparaffinization procedure should be
repeated.
# Stained
Tissue (+) Total # Notes 5. If tissue sections wash off the slide, slides should be checked to ensure that they are
Fallopian Tube 0 1 positively charged. Other possibilities that could have adverse affect on tissue adhesion
include insufficient drying of the tissue section on the slide prior to staining or fixation
Ureter 0 1
in formalin that was not properly neutral-buffered. Tissue thickness may also be a
Cervix 0 1
contributing factor.
Skeletal Muscle 0 1
Smooth Muscle 0 1 For corrective action, refer to the Instructions for Use section or contact Cell Marque customer
Skin 0 1
service.
Peripheral Nerve 0 1
Mesothelium 0 1 References
Fat 0 1
1. Jagirdar J. Application of immunohistochemistry to the diagnosis of primary and
Placenta 0 1
metastatic carcinoma to the lung. Arch Pathol Lab Med. 2008; 132:384-96.
This antibody stains normal tissues as indicated in literature. This antibody does not react with
colonic mucosa. 2. Bishop JA, et al. Napsin A and thyroid transcription factor-1 expression in carcinomas
of the lung, breast, pancreas, colon, kidney, thyroid, and malignant mesothelioma. Hum
Pathol. 2010; 41:20-5.
Disease Tissue Study
3. Rawlings ND and Salvesen GS. Handbook of Proteolytic Enzymes Volume 1. 3rd Edition.
# Stained Academic Press. 2013; p.69-71.
Tissue (+) Total # Notes
4. Brasch F, et al. Involvement of napsin A in the C- and N-terminal processing of surfactant
Lung adenocarcinoma 10 12
protein B in type-II pneumocytes of the human lung. J Biol Chem. 2003; 278: 49006-14.
Renal cell carcinoma 2 10
Breast invasive ductal carcinoma 0 14 5. Dejmek A, et al. Napsin A (TA02) is a useful alternative to thyroid transcription factor-1
Colorectal carcinoma 0 22 (TTF-1) for the identification of pulmonary adenocarcinoma cells in pleural effusions.
Diagn Cytopathol. 2007; 35:493-7.
Embryonal carcinoma 0 1
Lung Neuroendocrine Carcinoma 0 3 6. Inamura K, et al. Pulmonary adenocarcinomas with enteric differentiation: histologic and
Lung squamous carcinoma 0 13 immunohistochemical characteristics compared with metastatic colorectal cancers and
Melanoma 0 1 usual pulmonary adenocarcinomas. Am J Surg Pathol. 2005; 29:660-5.
Mesothelioma 0 3 7. Ye J, et al. Combination of Napsin A and TTF-1 immunohistochemistry helps in
Ovarian Serous Carcinoma 0 3 differentiating primary lung adenocarcinoma from metastatic carcinoma in the lung. Appl
Pancreatic carcinoma 0 5 Immunohistochem Mol Morphol. 2011; 19:313-17.
Prostate carcinoma 0 5 8. Mukhopadhyay S, et al. Subclassification of non-small cell lung carcinomas lacking
Transitional cell carcinoma 0 5 morphologic differentiation on biopsy specimens: Utility of an immunohistochemical
In house studies show that Napsin A (MRQ-60) mouse monoclonal antibody does not have panel containing TTF-1, napsin A, p63, and CK5/6. Am J Surg Pathol. 2011; 35:15-25.
cytoplasmic dot-like staining in colonic mucosa, which otherwise has been seen with rabbit
polyclonal Napsin A.
Disclaimers

Troubleshooting
*TWEEN is a registered trademark of Croda International PLC.
1. If the positive control exhibits weaker staining than expected, other positive controls
©2017 Sigma-Aldrich Co. LLC. All rights reserved. SIGMA-ALDRICH is a trademark of Sigma-
run during the same staining run should be checked to determine if it is because of the
Aldrich Co. LLC, registered in the US and other countries. Cell Marque, Trilogy, Declere, and
primary antibody or one of the common secondary reagents.
HiDef Detection are trademarks of Sigma-Aldrich Co. LLC or its affiliates.
2. If the positive control is negative, other positive controls used on the same run should
be checked to determine if the underlying cause relates to the primary antibody or one
of the common secondary reagents. Tissues may have been improperly collected, fixed www.cellmarque.com
or deparaffinized. The proper procedure should be followed for collection, storage, and
fixation. 6600 Sierra College Blvd. • Rocklin, CA 95677 USA • 916-746-8900
3. If excessive background staining occurs, high levels of endogenous biotin may be present. EMERGO EUROPE
A biotin blocking step should be included unless a biotin-free detection system is being Prinsessegracht 20, 2514 AP, The Hague, The Netherlands
used in which case any biotin present would not be a contributing factor to background
staining.

Napsin A (MRQ-60) Antibody

 CMC35229020 • EN Rev. 2.0 • p. 6

CM Template #2.2v1
Implemention date 22 Mar 2017

Napsin A (MRQ-60) Antibody