Intended Use
For In Vitro Diagnostic Use
Summary and Explanation
Cytokeratin 5 (58 kDa) is a high-molecular weight, basic type of cytokeratin expressed in basal, intermediate and superficial-cell layers of stratified epithelia as well as transitional epithelia, complex epithelia, mesothelial cells and Mesothelioma. Cytokeratin 6 (56 kD) is also a high-molecular weight, basic type cytokeratin expressed by proliferating squamous epithelium often paired with Cytokeratin 16.
CK 5 and 6 are positively seen in nearly 100% of Malignant Mesotheliomas and is rarely seen in Lung Adenocarcinomas. CK 5 and 6 can positively be seen in undifferentiated Large-cell Carcinoma as well as Squamous Carcinoma. Fewer than 10% of Carcinomas of the breast, colon, and prostate stain positively for this marker. CK 5 and 6 have also been used successfully as a myoepithelial cell marker in the prostate to determine malignancy.
Presentation
Anti-CK 5 & 6 is a cocktail of mouse monoclonal antibodies derived from cell culture supernatant that is concentrated, dialyzed, filter sterilized and diluted in buffer pH 7.5, containing BSA and sodium azide as a preservative.
Mohs IHC Procedure
Specimen Preparation of Mohs Frozen Tissues
- Embed the specimen in OCT inside a cryostat.
- Cut sections at 4-5 µm and mount on a positively charged glass slide such as the Bio SB Hydrophilic Plus Slides (BSB 7028) or in the lower third of the TintoDetector Cap Gap slides (BSB 7006).
- Air dry the slide at room temperature for 2 minutes and then incubate the slide at 60 °C for 3 minutes in an incubator or dry bath.
- Fix in 100% acetone or 10% NBF for 2 minutes at room temperature, Using 10% NBF produces better morphology if using Mohs ImmunoDigestor.
- For slides fixed in avetone, let it air dry. For slides fixed in NBF, rinse with distilled water and air dry.
Pretreatment of Mohs Frozen Tissues
- Transfer slides to ImmunoDNA Washer, TBST or PBST buffer.
- Perform PIER, proteolytic Digestion with Mohs ImmunoDigestor for 1 min. at room temperature.
- Rinse with ImmunoDNA Washer, TBST or PBST buffer.
IHC Detection PRocedure
- After PIER, transfer slides to ImmunoDNA washer and let it stand for 1-2 minutes.
- For manual staining, perform antibody incubation at ambient temperature. For automated staining methods, perform antibody incubation according to instrument manufacturer’s instructions.
- Wash slides with ImmunoDNA washer or DI water.
- Continue IHC detection protocol. Wash slides between each step with ImmunoDNA washer solution.
Abbreviated Mohs PolyDetector DAB HRP Brown or HRP Green Immunohistochemical Protocol
- Incubate slides with Peroxidase Blocker for 30 seconds
- Wash with Buffer (TBST or PBST)
- Incubate with Primary Antibody for 4 min
- Wash with Buffer (TBST or PBST)
- Incubate with HRP Label for 3 min
- Wash with Buffer (TBST or PBST)
- Prepare
- DAB Brown (1 drop of DAB Chromogen in 1ml of DAB Buffer; mix well)
- or HRP Green (1 drop of HRP Green Chromogen in 1 ml of HRP Green Buffer; mix well)
- Incubate with DAB or HRP Green for 2 min
- Wash with Buffer (TBST or PBST)
- Counterstain with Hematoxylin or Nuclear Fast Red for 30 seconds
- Wash with Buffer (TBST or PBST)
- Mount with AquaMounter or dehydrate the tissue with Fast ChromoProtector then mount with PermaMounter
Step
|
Mohs PolyDetector HRP Green or DAB Protocol |
| Peroxidase Blocker |
0.5 min. |
| Primary Antibody |
4 min. |
| 1st Step Detection |
3 min. |
| Substrate-Chromogen |
1-2 min. |
| Counterstain / Coverslip |
Varies |
This protocol can also be used with FFPE tissues retrieved with Citrate or EDTA.
