IHC of Cytokeratin CAM5.2 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 Anti-Cytokeratin CAM5.2 antibody has a primary reactivity with human keratin proteins that correspond to Moll’s peptides #7 and #8, Mr 48 and 52 kDa, respectively. Cytokeratin 7 and 8 are present in secretory epithelia of normal human tissue but not on stratified squamous epithelium.

Anti-Cytokeratin (CAM5.2) stains most epithelial-derived tissue, including liver, renal tubular epithelium, and Hepatocellular and Renal Cell Carcinomas. Anti-Cytokeratin (CAM 5.2) may not react with some Squamous Cell Carcinomas.

Antibody Type Mouse Monoclonal Clone CAM5.2
Isotype IgG2a/K Reactivity Paraffin, Frozen
Localization Cytoplasmic Control BCC, Merkel Cell Carcinoma, Mucinous Carcinoma, EMPD
Presentation Anti-Cytokeratin CAM5.2 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 3675 TintoFast Prediluted Ready-To-Use 3.0 ml
BSB 3676 TintoFast Prediluted Ready-To-Use 7.0 ml
BSB 3677 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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