Vector Laboratories
VectaStain Elite ABC Kit, Vector Laboratories
#PK-6101
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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 beta isoform in paraffin-embedded mouse ovary sections by immunohistochemistry, as part of studying the expression of the seven 14-3-3 isoforms. I selected the Avidin: Biotinylated enzyme Complex (ABC) technique because it relies on the high affinity of avidin for biotin and produces minimal background staining in absence of primary antibody. Immunohistochemical staining 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 follicular development. This study may help to elucidate isoform-specific protein interactions and functional roles of 14-3-3-beta in mammalian reproductive development.
Immunohistochemistry
4% paraformaldehyde-fixed, paraffin-embedded ovary and brain (positive control) tissue sections.
Tissue sections were incubated overnight at 4°C in a humidified chamber with rabbit anti-14-3-3 beta primary antibody (from the 14-3-3 isoform panel PAN017, AbD Serotec) diluted 1:600 in blocking buffer at dilutions recommended by the manufacturer and a prior study. The same dilutions were used for positive control sections. The rabbit serum used in matching negative control sections, were immunostained at a dilution identical to that used in corresponding sample sections for the isoform tested. Brain tissue sections were used as positive control.
Incubation of tissue sections for 20 min in PBS blocking buffer or 0.15% normal goat serum (VectaStain Elite ABC Kit, #PK-6101, Vector Laboratories).
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.
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.
Regions stained brown indicate presence of the 14-3-3 isoforms in contrast with regions counterstained blue. Standard high resolution microscopy was undertaken using Microsuite imaging software.
I showed by immunohistochemistry that the 14-3-3 beta isoform of 14-3-3 protein is detected in cells of the ovary. Follicles at various stages of development exhibit some common features of expression of the isoforms. Te beta isoform of 14-3-3 was detected, to varying extents, 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. For this isoform, 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. Several characteristic differences in expression of 14-3-3 beta protein isoform in different stages of follicular development were observed by immunohistochemical staining. Detection of 14-3-3β in zonae pellucidae of oocytes by immunohistochemical staining indicates secretion of some 14-3-3 proteins into the zonae and perivitelline space. Control ovary sections did not show brown staining, confirming specific localization by this method. Brain tissue sections were used as positive control for 14-3-3 beta.
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High specificity, low background compared to some fluorescence IHC.
The price may be a bit on the higher end.
Great product! Fast and convenient, producing robust yet reliable results.