TintoFast NGFR Antibody anti-NGFR immunohistochemistry IHC

TintoFast NGFR Antibody Immunohistochemistry on a Frozen Acetone-fixed Squamous Cell Carcinoma Tissue

Specification Sheets
Safety Data Sheet
Intended UseFor Mohs In Vitro Diagnostic Use
Summary and ExplanationTintoFast NGFR (Nerve Growth Factor Receptor), also termed p75 or CD271, is the low-affinity NGFR (LNGFR) which binds NGF and other neurotrophins, including BDNF, NT3 and NT4/5 with similar low-affinity. NGFR p75 is a 75 kD transmembrane glycoprotein that is mainly expressed in Schwann cells and neurons and in a variety of nonneuronal cells. NGFR p75 is necessary for
regulating neuronal growth,migration, differentiation and cell death during development of the central and peripheral nervous system. NGFR p75 plays a central role in the regulation of cell number by apoptosis in the developing CNS. During early development, activation of NGFR p75 by NGF induces apoptotic cell death in some neuronal cells, probably through activation of the sphingomyelinase/ceramide pathway, the ICE-like proteases and the JNK pathway. CD271 has recently been described as being expressed in mesenchymal stem cells (bone marrow stromal cells).TintoFast NGFR is expressed not only in sympathetic and sensory neurons, but also in various neural crest cell or tumor derivatives such as melanocytes, Melanomas, Neuroblastomas, Pheochromocytomas, Neurofibromas, and neurotized nevi (Type C melanocytes). It is now apparent that expression of NGFR is ubiquitous and not limited to the nervous system. Studies in Prostate and Urothelial Cancer suggest that NGFR may act as a tumor suppressor, negatively regulating cell growth and proliferation. NGFR labels the myoepithelial cells of breast ducts and intralobular fibroblasts of breast ducts and, thus, aids in the diagnosis of malignancy in the breast.
Antibody TypeMouse MonoclonalCloneBSB-18
IsotypeIgG1ReactivityParaffin, Frozen
LocalizationCytoplasmicControlBrain, Breast, Prostate, Neuroblastoma, CNS Tumor
PresentationTintoFast NGFR 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 3696 – 3TintoFast PrediluteReady-To-Use3.0 ml
BSB 3696 – 7TintoFast PrediluteReady-To-Use7.0 ml
BSB 3696 – 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.