Microphthalmia Antibody Staining Protocol for Immunohistochemistry

 

Description: Mi is a basic helix-loop-helix-leucin zipper (b-HLH-ZIP) transtripotion factor implicated in pigmentation, mast cells and bone development. The mutation of Mi causes Waardenburg Syndrome type II in humans. In mice, a profound loss of pigmented cells in the skin eye and inner ear results, as well as osteopetrosis and defects in natural killer and mast cells. There are two known isoforms of Mi differing by 66 amino acids at the NH2 terminus. Shorter forms are expressed in melanocytes and run as two bands at 52kDa and 56kDa, while the longer Mi form runs as a cluster of bands at 60-70kDa in osteoclasts and in B16 melanoma cells (but not other melanoma cell lines), as well as mast cells and heart.

 

Primary Antibody

Name: Microphthalmia (Transcription Factor) (MiTF) Ab-1 Antibody

Clone: MiTF, Mouse anti-Human

Supplier: Lab Vision

Catalog Number: MS-771-P

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

Counterstain
Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

Results:
Staining Pattern: Nuclear
Images: Search image

Additional Information:
Tissue Type: Melanoma, 501 Mel Cells, Bone marrow
Fixation: Formalin fixed paraffin sections
Positive Control: Melanoma, 501 Mel Cells
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

 

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