tintofast cytokeratin 5 & 6 ck5 ck6 anti-ck5 anti-ck6 antibody immunohistochemistry ihc

TintoFast Cytokeratin 5 & 6 Antibody Immunohistochemistry on a Frozen Acetone-Fixed Melanoma Tissue

Specification Sheets
Safety Data Sheet
Intended UseFor Mohs 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.

TintoFast Cytokeratin 5 & 6 are positively seen in nearly 100% of Malignant Mesotheliomas and is rarely seen in Lung Adenocarcinomas. TintoFast Cytokeratin 5 & 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.

Antibody TypeMouse MonoclonalCloneD5/16B4
IsotypeIgG1ReactivityParaffin, Frozen
PresentationAnti-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.
Catalog No.Antibody TypeDilutionVolume/QTY
BSB 3666TintoFast PredilutedReady-To-Use3.0 ml
BSB 3667TintoFast PredilutedReady-To-Use7.0 ml
BSB 3668TintoFast PredilutedReady-To-Use15.0 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 10% NBF 10% produces better morphology if using Bio SB Mohs ImmunoDigestor.
  5. For slides fixed in acetone, let it air dry. For slides in NBF, rinse with distilled water and air dry.

Pretreatment of Mohs Frozen Tissues

  1. Transfer slides to ImmunoDNA Washer, TBST or PBST buffer.
  2. Perform PIER, proteolytic Digestion with Mohs ImmunoDigestor for 1 min. at room temperature.
  3. Rinse with ImmunoDNA Washer, TBST or PBST buffer.

IHC Detection Procedure

  1. After PIER, transfer slides to ImmunoDNA washer and let it stand for 1-2 minutes.
  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 or DI water.
  4. Continue IHC detection protocol. Wash slides between each step with ImmunoDNA washer solution.

Abbreviated Mohs PolyDetector DAB HRP Brown of HRP Green Immunohistochemical Protocol

  1. Incubate slides with Peroxidase Blocker for 30 seconds
  2. Wash with Buffer (TBST or PBST)
  3. Incubate with Primary Antibody for 4 min
  4. Wash with Buffer (TBST or PBST)
  5. Incubate with HRP Label for 3 min
  6. Wash with Buffer (TBST or PBST)
  7. 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)
  8. Incubate with DAB or HRP Green for 2 min
  9. Wash with Buffer (TBST or PBST)
  10. Counterstain with Hematoxylin or Nuclear Fast Red for 30 seconds
  11. Wash with Buffer (TBST or PBST)
  12. Mount with AquaMounter or dehydrate the tissue with Fast ChromoProtector then mount with PermaMounter

StepMohs PolyDetector HRP Green or DAB Protocol
Peroxidase Blocker30 seconds
Primary Antibody4 min.
1st Step Detection (HRP Label)3 min.
Substrate-Chromogen1-2 min.
Counterstain / CoverslipVaries

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