Isolectin IB4 Biotin Conjugates Antibody Staining Protocol for Immunohistochemistry

 

Description: Isolectin IB4 is a 114 kDa glycoprotein and part of a family of five tetrameric type I isolectins (IA4, IA3B, IA2B2, IAB3, and IB4) isolated from the seeds of the tropical African legume Griffonia simplicifolia (formerly Bandeiraea simplicifolia). The A and B subunits comprising the family members are very similar, differing in amino acid sequence only at the N-terminus. However, the subunits display different binding specificities; the A subunit prefers N-acetyld-galactosamine end groups while the B subunit is selective for terminal α-d-galactosyl residues.

 

Isolectin IB4 specifically agglutinates blood group B erythrocytes and was originally employed
for this purpose.2 Subsequent work has shown that isolectin IB4 is cytotoxic to several normal
and tumor cell types 3 and has particularly strong affinity for brain microglial and perivascular
cells.4 It has also been particularly valuable as a histochemical and flow cytometric probe for
specifically labeling endothelial cells in a number of species.5,6 Isolectin IB4 has been used effectively for tracing central and peripheral neuronal pathways following local injections,7,8 as
well as for labeling stimulated murine macrophages,9 bovine thyroid cells,10 various murine
cell types,11,12 laminin,13 and thyroglobulin.14 Since the applications of isolectin IB4 are varied, researchers should consult the primary literature for protocol information.

 

Primary Antibody

Name: Isolectin IB4 Biotin Conjugates Antibody

Clone: IB4

Supplier: Invitrogen

Catalog Number: I21414

Dilution: 1:2000 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: No pretreatment is needed
Buffer/pH value: N/A
Heat/Cool Temperature: N/A
Heat/Cool Time: N/A

Detection Methods
Standard Method: ABC Method or LSAB Method (Note: since this antibody is biotin conjugated, secondary antibody step should be omitted, serum blocking step prior to primary antibody incubation is not needed neither)
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: Membrane/cytoplasmic of blood vessel wall
Images: Search image

Additional Information:
Tissue Type: Heart
Fixation: Formalin fixed paraffin sections. Not tested on frozen sections.
Positive Control: Heart
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

 

References: