IHC of MART-1 on a Frozen Acetone-Fixed Melanoma Tissue

Specification Sheets
Safety Data Sheet

SDS

Intended Use For Mohs In Vitro Diagnostic Use
Summary and Explanation

MART-1/Melan-A is a putative 18 kDa transmembrane protein consisting of 118 amino acids. It has a single transmembrane domain. MART-1/ Melan-A is a protein antigen found on melanocytes. Antibodies against this antigen are used to recognize cells of melanocytic differentiation, useful for the diagnosis of Melanoma.  The same name is used to refer to the gene which codes for this antigen.

The MART-1/Melan-A antigen is specific for the melanocyte lineage found in normal skin, retina, and melanocytes, but not in other normal tissues. It is thus useful as a marker for Melanocytic Tumors, with the caveat that it is normally found in benign nevi as well. This antibody is very useful in establishing the diagnosis of Metastatic Melanomas.

Antibody Type Mouse Monoclonal Clone A103
Isotype IgG1 Reactivity Paraffin, Frozen
Localization Cytoplasmic Control Skin, Melanoma
Presentation Anti – MART-1 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 3651 TintoFast Prediluted Ready-To-Use 3.0 ml
BSB 3652 TintoFast Prediluted Ready-To-Use 7.0 ml
BSB 3653 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 Plus 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 for 2 minutes at room temperature.
  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 or 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, or TBST or PBST Buffer or DI water.
  4. Continue IHC detection protocol. Wash slides between each step with ImmunoDNA washer or Tris or PBS Buffer solution.

Abbreviated Mohs Immunohistochemical Protocol

  1. Incubate slides with Peroxidase Blocker for 30 seconds
  2. Wash with Buffer (TBST or PBST)
  3. Incubate Mart-1 for 4 min
  4. Wash with Buffer (TBST or PBST) for
  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 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 MART-1, we recommend using Mohs Frozen sections fixed 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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