Application Of The 3,3'-Diaminobenzidine (DAB) Substrate In ABC Immunohistochemical Staining

August 03, 2016

Animal Sciences
Purdue University
Postdoctoral Fellow

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Company:

Vector Laboratories

Product Name:

3,3'-Diaminobenzidine (DAB) peroxidase substrate kit, Vector Laboratories

Catalog Number:

SK-4100

Browse Similar Products: DAB Immunohistochemistry Antibodies

The 14-3-3 (YWHA) proteins are a highly conserved, ubiquitously expressed family of proteins controlling important cellular processes, with seven known mammalian isoforms – beta, gamma, epsilon, zeta, eta, tau/theta and sigma. I examined the expression of 14-3-3 beta isoform in paraffin-embedded mouse ovary sections by ABC immunohistochemical staining, as part of studying the expression of the seven 14-3-3 isoforms. I selected the DAB substrate because it is known to generate distinct brown color upon reaction with the VectaStain Elite ABC reagent used in this method, and produces minimal background staining in absence of primary antibody. ABC immunohistochemistry detected 14-3-3 beta in ovarian follicles, with some similarities as well as notable differences in relative amounts, localizations and patterns of expression in multiple cell types at various stages of follicuogenesis. This study may help to elucidate isoform-specific protein interactions and functional roles of 14-3-3-beta in mammalian reproductive development.

Experimental Design and Results Summary

Applications

Immunohistochemistry

Sample

Formaldehyde-fixed, paraffin-embedded mouse tissue sections

Primary Incubation

Incubation overnight at 4°C in a humidified chamber with rabbit anti-14-3-3 beta primary antibody (included in 14-3-3 isoform panel PAN017, AbD Serotec) diluted 1:600 in blocking buffer at dilutions recommended by the manufacturer and a prior study

Blocking Agent

Endogenous peroxidase is blocked by incubation of tissue sections in 0.3% hydrogen peroxide for 30 min. This is followed by incubation for 20 min in PBS blocking buffer or 0.15% normal goat serum (VectaStain Elite ABC Kit, #PK-6101, Vector Laboratories)

Secondary Incubation

Incubation for 30 min in biotinylated secondary antibody (VectaStain Elite ABC Kit, Vector Laboratories) diluted in blocking buffer as per manufacturer's instructions

Tertiary Incubation

Incubation of tissues for 30 min in VectaStain Elite ABC Reagent (prepared according to the manufacturer's instructions; Vector Laboratories). DAB substrate is prepared using Vector Laboratories DAB peroxidase substrate Kit (SK-4100) according to instructions specified in kit. Tissues are treated with DAB substrate for 3-4 min until development of optimum brown color, rinsed with tap water and counter-stained with Hematoxylin.

Detection

Standard detection using a high-resolution microscope and Multisuite imaging software

Results Summary

The 14-3-3 beta isoform was detected in the oocyte and cumulus cells surrounding the oocyte, mural granulosa cells, theca interna and theca externa of all follicular stages examined, as well as in cells of the corpus luteum. In each of the follicular stages studied, the isoform appears to be expressed in the cytoplasm of the oocytes and to some extent in the corresponding germinal vesicles. Staining appeared more intense in the cytoplasm than in the nuclei of somatic cells in granulosa and theca layers as well as in cells of corpora lutea. Cells within corpora lutea were also found to have relatively higher amounts of expression of all of the isoforms as compared to surrounding interstitial cells.

Additional Notes

None

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Summary

The Good

The Vector Labs DAB substrate is sensitive, reliable and fast.

The Bad

Sometimes, quantification of the intensity of the brown color developed may be needed to validate qualitative results. Also, good controls are essential to rule out false positive results of the color generation.

The Bottom Line

Good substrate for immunohistochemistry by the avidin-biotin conjugated method.

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