Effectiveness Of Rabbit Anti-14-3-3 Sigma To Detect The Protein In Mouse Ovaries By Immunohistochemistry

August 11, 2016

Animal Sciences
Purdue University
Postdoctoral Fellow

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Company:

Bio-Rad (Formerly AbD Serotec)

Product Name:

Rabbit anti 14-3-3 sigma

Catalog Number:

AHP1050

The 14-3-3 (YWHA) proteins are a highly conserved, ubiquitously expressed family of proteins controlling important cellular processes, with seven known mammalian isoforms – β, γ, ε, ζ, η, τ and, σ. I examined the expression of 14-3-3 sigma isoform in paraffin-embedded mouse ovary sections by immunohistochemistry. In order to pursue this project, I selected the rabbit anti-14-3-3 sigma (AHP1050, AbD Serotec) as I had effectively used this antibody to report the expression of this protein in mouse oocytes and eggs (De et al., 2012). Immunohistochemical staining detected 14-3-3 sigma in mouse 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 follicular development. This study may help to elucidate isoform-specific protein interactions and functional roles of 14-3-3 sigma 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 sigma primary antibody diluted 1:100 in blocking buffer

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 of tissue sections 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 tissue sections for 30 min in VectaStain Elite ABC Reagent (prepared according to the manufacturer's instructions; Vector Laboratories). DAB substrate was prepared using Vector Laboratories DAB peroxidase substrate Kit (SK-4100) according to instructions specified in kit. Tissues were 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

Immunohistochemical staining detected 14-3-3 sigma within mouse 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 follicular development as illustrated in the figure provided.

Additional Notes

None

Image Gallery

Summary

The Good

Specificity seems high, and the antibody comes with a standardized ready-to-use concentration, and has a high affinity for the 14-3-3 isoform.

The Bad

The 14-3-3 sigma isoform is not present in all tissues; so appropriate controls may be needed. The dilution and specificity of the antibody may need to be double checked depending on the cell/tissue used, and the application.

The Bottom Line

Good product, ready-to-use and comes with multiple published applications.

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