CD15 Antibody Staining Protocol for Immunohistochemistry

Description: CD15 recognizes a human myelomonocytic antigen. The CD15 antigen is present on greater than 95% of mature peripheral blood eosinophils and neutrophils and is present at low density on circulating monocytes. In lymphoid tissue, CD15 reacts with Reed-Sternberg cells of Hodgkin's disease and with granulocytes; however, CD15 reacts with few tissue macrophages and does not react with dendritic cells.

Primary Antibody

Name: CD15 Antibody

Clone: C3D-1, Mouse anti-Human

Supplier: Dako

Catalog Number: M0733

Dilution: 1:20 using IHC-Tek^TM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed.

Incubation Time/Temp: 60min/room temperature

Antigen Retrieval

Device: IHC-Tek^TM Epitope Retrieval Steamer Set (Cat# IW-1102)

Buffer/pH value: IHC-Tek^TM 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: Gill's Hematoxylin or Mayer's Hematoxylin

Staining Time: 30 seconds

Results:

Staining Pattern: Membrane/cytoplasmic

Images: Search image

Additional Information:

Tissue Type: Tonsil

Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections

Positive Control: Tonsil

Negative Control: Omit primary antibody, isotype control or 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

Notes:

1. There is no need to perform antigen retrieval for acetone fixed frozen sections

2. CD15 (Clone MMA) from Becton Dickinson (Cat# 347420) also works well at 1:20 dilution

References: