Melanosome Antibody Staining Protocol for Immunohistochemistry


Description: Anti-melanosome, HMB45 has been shown to react with a 10 kDa segment of a neuraminidase-sensitive sialylated glycoconjugate present in pre- and earlystage (immature) melanosomes.The presence of the antigen indicates active melanosome formation and thus melanocytic differentiation. It is also expressed in normal fetal melanocytes, but not in normal resting adult melanocytes, regardless of the degree of pigmentation. Upon activation, adult melanocytes can re-express the HMB45-defined antigen (as it is expressed in fetal melanocytes). Such melanocytes are activated by a variety of stimuli. For example, HMB45-positive cells have been detected in tissue overlying or adjacent to granulation tissue, hemangiomas, vessel-rich tumor stroma, and basal cell carcinoma. Hair follicles stain occasionally due to associated stimulated melanocytes. Positive HMB45 staining has not been observed with melanocytes in lentigines or overlying fibroblastic proliferations such as keloids, dermatofibromas and old fibrotic hemangiomas.8 Nonmelanocytic normal tissues do not react with the HMB45 antibody.


Primary Antibody

Name: Melanosome Antibody

Clone: HMB45, Mouse anti-Human

Supplier: Dako

Catalog Number: M0634

Dilution: 1:100 using IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed.

Incubation Time/Temp: 60 min/room temperature

Antigen Retrieval
Device: IHC-TekTM Epitope Retrieval Steamer Set (Cat# IW-1102)
Buffer/pH value: IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100)
Heat/Cool Temperature: 95-100 ºC/room temperature
Heat/Cool Time: 20 minutes/20 minutes

Detection Methods
Standard Method: ABC Method or LSAB Method
Enhanced Method: Polymeric Methods

Chromogen Substrate
Reagent: DAB
Incubation Time/Temperature: 1-3 minutes/room temperature

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

Staining Pattern: Cytoplasmic
Images: Search image

Additional Information:
Tissue Type: Skin
Fixation: Formalin fixed paraffin sections, acetone-fixed frozen sections, and cell smears.
Positive Control: Melanocytes (fetal and subset, melanocytes containing immature melanosomes), retinal pigment epithelia (prenatal and infantile).
Negative Control: Omit primary antibody, isotype control, absorption control
Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary