Intended Use
For 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%).
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.
Mohs IHC Procedure
Specimen Preparation of Mohs Frozen Tissues
- Embed the specimen in OCT inside a cryostat.
- 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).
- 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.
- Fix in 100% acetone for 2 minutes at room temperature.
Pretreatment of Mohs Frozen Tissues
- Preheat the TintoDetector Incubator to 110°C.
- Place TintoDetector Cap Gap slides (BSB 7006) face to face and insert them into the TintoDetector Slide Holder (BSB 7003).
- Submerge slides in ImmunoDNA Retriever with EDTA to draw up enough solution by capillary action to cover the tissues.
- Heat the slides in a preheated TintoDetector Incubator for 3 minutes.
- Transfer slides to room temperature and cool off for 1 min.
Mohs IHC Detection
- After HIER, transfer slides to ImmunoDNA washer and let it stand for 1-2 minutes.
- For manual staining, perform antibody incubation at ambient temperature. For automated staining methods, perform antibody incubation according to instrument manufacturer’s instructions.
- Wash slides with ImmunoDNA washer or DI water.
- Continue IHC detection protocol. Wash slides between each step with ImmunoDNA washer solution.
Abbreviated Mohs PolyDetector Plus DAB HRP Brown or HRP Green Immunohistochemical Protocol
- Incubate with Primary Antibody for 5 min
- Wash with Buffer (TBST or PBST)
- Incubate with M/R link for 4 min
- Wash with Buffer (TBST or PBST)
- HRP Label for 4 min.
- Wash with Buffer (TBST or PBST)
- 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)
- Incubate with DAB or HRP Green for 1-2 min
- Wash with Buffer (TBST or PBST)
- Counterstain with Hematoxylin or Nuclear Fast Red for 30 seconds
- Wash with Buffer (TBST or PBST)
- Mount with AquaMounter or dehydrate the tissue with Fast ChromoProtector then mount with PermaMounter
Step
|
Mohs PolyDetector Plus HRP Green or DAB 20 Minute Protocol |
| HIER |
3 min. |
| Primary Antibody |
5 min. |
| 1st Step Detection |
4 min. |
| 2nd Step Detection |
4 min. |
| Substrate-Chromogen |
1-2 min. |
| Counterstain / Coverslip |
Varies |
This protocol can also be used with FFPE tissues retrieved with Citrate or EDTA.
