IHC of Cytokeratin AE1/AE3 on a Frozen NBF 10%-Fixed Basal Cell Carcinoma Tissue

Specification Sheets
Safety Data Sheet

SDS

Intended Use For Mohs In Vitro Diagnostic Use
Summary and Explanation Cytokeratins are intermediate-filament keratins found in the intracytoplasmic cytoskeleton of epithelial tissue. There are two types of cytokeratins: the low-weight, acidic Type I cytokeratins and the high-weight, basic or neutral Type II cytokeratins. Cytokeratins are usually found in pairs comprising a Type I cytokeratin and a Type II cytokeratin. Expression of these cytokeratins is frequently organ or tissue-specific.

Cytokeratin cocktail AE1/AE3 is well suited to distinguish Epithelial Carcinoma from Non-epithelial malignancies and is used to aid Epithelial Tumor classification.  This antibody has been used to characterize the source of various neoplasms and to study the distribution of keratin-containing cells in epithelia during normal development and during the development of epithelial neoplasms.  This antibody stains cytokeratins present in normal and abnormal human tissues. This antibody has shown high sensitivity and specificity in recognizing epithelial cells of neoplastic origin.

Antibody Type Mouse Monoclonal Clone AE1/AE3
Isotype IgG1 Reactivity Paraffin, Frozen
Localization Cytoplasmic Control Skin, SCC, BCC
Presentation Anti – Cytokeratin AE1/AE3 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.
Availability
Catalog No. Antibody Type Dilution Volume/QTY
BSB 3663 TintoFast Prediluted Ready-To-Use 3.0 ml
BSB 3664 TintoFast Prediluted Ready-To-Use 7.0 ml
BSB 3665 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.

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