IHC of Cytokeratin 7 on a Frozen NBF-10%-Fixed Extramammary Paget’s Disease Tissue

Specification Sheets
Safety Data Sheet

SDS

Intended Use For Mohs In Vitro Diagnostic Use
Summary and Explanation Cytokeratin 7 (CK7) reacts with proteins that are found in most ductal, glandular and transitional epithelium of the urinary tract and bile duct epithelial cells. CK7 distinguishes between lung and breast epithelium that stain positive, and colon and prostate epithelial cells that are negative.

This antibody also reacts with many benign and malignant epithelial lesions (e.g., Adenocarcinomas of the ovary, breast and lung). Further, in frozen sections, the antibody has been shown to label the rete epithelium in the testis, epididymis epithelium, and the surface epithelium of the stomach and duodenum. Transitional Cell Carcinomas are positive and Prostate Cancers are negative. This antibody does not recognize intermediate filament proteins, nor does it recognize non-epithelial tissues such as blood vessels, connective tissue, etc.

Antibody Type Mouse Monoclonal Clone OV-TL 12/30
Isotype IgG1/K Reactivity Paraffin, Frozen
Localization Cytoplasmic Control TCC, EMPD
Presentation Anti – Cytokeratin 7 is a mouse monoclonal antibody 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.
Availability
Catalog No. Antibody Type Dilution Volume/QTY
BSB 3669 TintoFast Prediluted Ready-To-Use 3.0 ml
BSB 3670 TintoFast Prediluted Ready-To-Use 7.0 ml
BSB 3671 TintoFast Prediluted Ready-To-Use 15.0 ml

Mohs IHC Procedure

Specimen Preparation of Mohs Frozen Tissues

  1. Embed the specimen in OCT inside a cryostat.
  2. 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).
  3. 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.
  4. Fix in 100% acetone or 10% NBF for 2 minutes at room temperature. Using NBF 10% produces better morphology.
  5. Rinse with distilled water and air dry the slides for another 2 minutes at room temperature.

IHC Detection Procedure

  1. Transfer slides to ImmunoDNA washer, TBST or PBST buffer.
  2. For manual staining, perform antibody incubation at ambient temperature. For automated staining methods, perform antibody incubation according to instrument manufacturer’s instructions.
  3. Wash slides with ImmunoDNA washer, TBST, PBST or DI water.
  4. Continue IHC detection protocol. Wash slides between each step with ImmunoDNA washer, Tris or PBS Buffer solution.

Abbreviated Mohs Immunohistochemical Protocol

  1. Do PIER, proteolytic Digestion, with MohsImmunoDigestor for 1 min
  2. Wash with Buffer (TBST or PBST)
  3. Incubate slides with Peroxidase Blocker for 30 seconds
  4. Wash with Buffer (TBST or PBST)
  5. Incubate CK for 4 min
  6. Wash with Buffer (TBST or PBST)
  7. Incubate with HRP Label for 3 min.
  8. Wash with Buffer (TBST or PBST)
  9. Prepare
    • DAB  Brown (1 drop of DAB chromogen in 1 ml of DAB Buffer; mix well)
    • or HRP Green (1 drop of HRP Green chromogen in 1 ml of HRP Green Buffer, mix well)
  1. Incubate with DAB or HRP Green for 2 min
  2. Wash with Buffer (TBST or PBST)
  3. Counterstain with Hematoxylin or Nuclear Fast Red for 30 seconds
  4. Wash with Buffer (TBST or PBST)
  5. Mount with AquaMounter or PermaMounter

For best results with the IHC of Cytokeratin’s, we recommend using Mohs Frozen sections fixed NBF10% for 2 min., then PIER with Mohs ImmunoDigestor at room tepmarature for 1 min.with acetone for 2 min.

Step Mohs PolyDetector HRP Green 5 min Protocol Mohs PolyDetector HRP Green 10 min Protocol
Peroxidase Blocker 0.5 min. 0.5 min.
Primary Antibody 2 min 4 min.
1st Step Detection 1 min 3 min.
Substrate-Chromogen 1 min 2 min.
Counterstain / Coverslip 0.5 min 0.5 min.

This protocol can also be used with FFPE Tissues retrieved with Citrate or EDTA.

Related Products