tintofast ema anti-ema antibody immunohistochemistry ihc

TintoFast EMA Antibody Immunohistochemistry on a Frozen Acetone-fixed Melanoma Tissue

Specification Sheets
Safety Data Sheet
Intended UseFor Mohs In Vitro Diagnostic Use
Summary and ExplanationTintoFast EMA (Epithelial Membrane Antigen) antibody is a mucin-like glycoprotein, shown to be useful as a pan-epithelial marker for detecting early metastatic loci of carcinoma in the bone marrow or liver. It stains normal and neoplastic cells from various tissues, including mammary epithelium, sweat glands and squamous epithelium. Hepatocellular Carcinoma, Adrenal Carcinoma and Embryonal Carcinomas are consistently EMA negative, so keratin positivity with negative EMA favors one of these tumors. EMA is frequently positive in meningioma, which can be useful when distinguishing it from other intracranial neoplasms. The absence of EMA can also be of value since negative EMA is characteristic of some tumors including Adrenal Carcinoma, Seminomas, Paraganglioma and Hepatoma.
Antibody TypeMouse MonoclonalCloneE29
IsotypeIgG2a/KReactivityParaffin, Frozen
LocalizationCytoplasmic, MembranousControlBreast, Skin, Colon, Kidney, Cervix
PresentationTintoFast EMA 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 TypeDilutionVolume/QTY
BSB 3692-3TintoFast PrediluteReady-To-Use3.0 ml
BSB 3692-7TintoFast PrediluteReady-To-Use7.0 ml
BSB-3692-15TintoFast PrediluteReady-To-Use15.0 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 and let the slide air dry.

Pretreatment of Mohs Frozen Tissues

  1. Preheat the TintoDetector Incubator to 110 °C.
  2. Place TintoDetector Cap Gap slides (BSB 7006) face to face and insert them into the TintoDetector Slide Holder (BSB 7003).
  3. Submerge slides in ImmunoDNA Retriever with EDTA to draw up enough solution by capillary action to cover the tissues.
  4. Heat the slides in a preheated TintoDetector Incubator for 3 minutes.
  5. Transfer slides to room temperature and cool off for 1 min.

Mohs IHC Detection

  1. After HIER, transfer slides to ImmunoDNA washer and let it stand for 1-2 minutes.
  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 DI water.
  4. Continue IHC detection protocol. Wash slides between each step with ImmunoDNA washer solution.

Abbreviated Mohs PolyDetector Plus DAB HRP Brown of HRP Green Immunohistochemical Protocol

  1. Incubate with Primary Antibody for 5 min
  2. Wash with Buffer (TBST or PBST)
  3. Incubate with M/R link for 4 min
  4. Wash with Buffer (TBST or PBST)
  5. HRP Label for 4 min.
  6. Wash with Buffer (TBST or PBST)
  7. 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)
  8. Incubate with DAB or HRP Green for 1-2 min
  9. Wash with Buffer (TBST or PBST)
  10. Counterstain with Hematoxylin or Nuclear Fast Red for 30 seconds
  11. Wash with Buffer (TBST or PBST)
  12. Mount with AquaMounter or dehydrate the tissue with Fast ChromoProtector then mount with PermaMounter

StepMohs PolyDetector HRP Green or DAB 20 Minute Protocol
HIER3 min.
Primary Antibody5 min.
1st Step Detection4 min.
2nd Step Detection4 min.
Substrate-Chromogen1-2 min.
Counterstain / CoverslipVaries

This protocol can also be used with FFPE Tissues retrieved with Citrate or EDTA.