IHC of MiTF on a Frozen Acetone-Fixed Melanoma Tissue

Specification Sheets
Safety Data Sheet

SDS

Intended Use For Mohs In Vitro Diagnostic Use
Summary and Explanation

Microphthalmia-associated Transcription Factor (MiTF) is a basic helix-loop-helix leucine zipper transcription factor involved in melanocyte and osteoclast development. Mutations in MiTF cause auditory pigmentary syndromes, such as Waardenburg Syndrome Type II, Type IIa and Tietz Syndrome in humans. There are two known isoforms of MiTF differing by 66 amino acids at the NH2 terminus. Shorter forms are expressed in melanocytes and run as two bands at 52 kDa and 56 kDa, while the longer Mi form runs as a cluster of bands at 60-70 kDa in osteoclasts and in B16 Melonoma cells (but not other Melanoma cell lines), as well as mast cells and heart cells. MiTF plays a critical role in the differentiation of various cell types such as neural crest-derived melanocytes, mast cells, osteoclasts and optic cup-derived retinal pigment epithelium.

This antibody recognizes serine phosphorylated and non-phosphorylated melanocytic isoforms of microphthalmia. It is useful in identifying Malignant Melanoma, and distinguishing mast cell lesions from lesions of myeloid derivation. A relatively rare class of tumors known as PEComas (tumors showing perivascular epithelioid cell differentiation) express MiTF in a high percentage of cases (~90%).

Antibody Type Mouse Monoclonal Clone C5/D5
Isotype IgG1/K Reactivity Paraffin, Frozen
Localization Nuclear Control Skin, Melanoma
Presentation Anti-MiTF is cocktail of two mouse monoclonal antibodies derived from cell culture supernatant that are 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 3657 TintoFast Prediluted Ready-To-Use 3.0 ml
BSB 3658 TintoFast Prediluted Ready-To-Use 7.0 ml
BSB 3659 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 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, 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 HIER with Citrate in Pressure Cooker (100 – 121 °C) for 5 min. Cool off
  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 Primary Antibody 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 we recommend using Mohs Frozen sections fixed NBF10% for 2 min., then HIER with Citrate for 5 min. at 110 – 121 °C.

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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