Monday, October 29, 2012

OPC Markers!

Effective Oligodendrocyte, Oligodendroglial Oligodendrocyte Lineage Markers are important for determining the differentiate state of Oligodendrocyte Precursor Cells. This is important for the study of de and re-myelination of neurons and the discovery of potential therapeutic targets for diseases like MS and ALS.

Here researchers use our Olig2 antibody to study the differentiation state of Fetal Human Oligodendrocyte Progenitor Cells: Crystal R. McClain, Fraser J. Sim and Steven A. Goldman. Pleiotrophin Suppression of Receptor Protein Tyrosine Phosphatase-β/ζ Maintains the Self-Renewal Competence of Fetal Human Oligodendrocyte Progenitor Cells. The Journal of Neuroscience, 24 October 2012, 32(43): 15066-15075; doi: 10.1523/​JNEUROSCI.1320-12.2012.
Abstract: Oligodendrocyte progenitor cells (OPCs) persist in human white matter, yet the mechanisms by which they are maintained in an undifferentiated state are unknown. Human OPCs differentially express protein tyrosine phosphatase receptor β/ζ (PTPRZ1) and its inhibitory ligand, pleiotrophin, suggesting the maintenance of an autocrine loop by which PTPRZ1 activity is tonically suppressed. PTPRZ1 constitutively promotes the tyrosine dephosphorylation of β-catenin and, thus, β-catenin participation in T cell factor (TCF)-mediated transcription. Using CD140a/PDGFRα-based fluorescence-activated cell sorting to isolate fetal OPCs from the fetal brain at gestational ages 16–22 weeks, we asked whether pleiotrophin modulated the expansion of OPCs and, if so, whether this was effected through the serial engagement of PTPRZ1 and β-catenin-dependent signals, such as TCF-mediated transcription. Lentiviral shRNAi knockdown of PTPRZ1 induced TCF-mediated transcription and substantially augmented GSK3β inhibition-induced TCF-reporter luciferase expression, suggesting dual regulation of β-catenin and the importance of PTPRZ1 as a tonic brake upon TCF-dependent transcription. Pharmacological inhibition of GSK3β triggered substrate detachment and initiated sphere formation, yet had no effect on either proliferation or net cell number. In contrast, pleiotrophin strongly potentiated the proliferation of CD140a+-sorted OPCs, as did PTPRZ1 knockdown, which significantly increased the total number of population doublings exhibited by OPCs before mitotic senescence. These observations suggest that pleiotrophin inhibition of PTPRZ1 contributes to the homeostatic self-renewal of OPCs and that this process is mediated by the tonic activation of β-catenin/TCF-dependent transcription.

Images: To verify that GSK3β inhibition was effecting TCF activation through altering localization of β-catenin, the Wnt signaling intermediate, β-catenin, was localized by confocal imaging in OPCs, validated as such by their coexpression of Olig2.

Marker Options:
NameCatalog #TypeSpeciesApplicationsSizePrice
CNPaseCH23013Chicken IgYH; MICC; IHC100 ul$89
Caspr2SP15104Sheep IgGH; MIHC; WB; E100 ug$365
HSP105MO20028Mouse IgGH; M; RIHC; WB100 ul$155
MAG/Siglec 4aGT15152Goat IgGRIHC; WB; E100 ug$365
MOGGT15141Goat IgGHIHC; WB; E100 ug$365
Mash1GT15216Goat IgGMIHC; WB; E100 ug$365
Mash1MO15048Rat IgGH; MICC; WB; E100 ug$255
NOGO ReceptorGT15154Goat IgGHIHC; WB; E100 ug$365
OMgpGT15200Goat IgGHWB; E100 ug$365
Olig1RA14141Rabbit IgGRIHC100 ul
100 ul @ 1mg/ml
Olig1,2,3MO15059Mouse IgGH; RIHC100 ug$305
Olig2GT15132Goat IgGH; MIHC; WB; E100 ug$365
Olig2RA25081Rabbit IgGH; M; RICC; IHC; WB; IP100 ul$395
Oligodendrocyte Marker O1MO15001Mouse IgMH; M; RIHC; FC50 ug$215
Oligodendrocyte Marker O4MO15002Mouse IgMC; H; M; RIHC50 ug$215
Oligodendrocyte Marker O4-Phycoerythrin LabeledFC15013Mouse IgMHFC100 Tests$305
PDGF R Alpha/CD140AGT15150Goat IgGMIHC; WB; E100 ug$365

Sunday, October 21, 2012

P2X3 Receptor and CGRP Antibodies Immunostaining

Dr. Alfredo Ribeiro-da-Silva, McGill University, is a serial publisher of studies using our pain and inflammation research antibodies.

Here, use of our rabbit anti-CGRP and guinea pig anti-P2X3 is referenced. Please note the high titer of these antibodies (dilution is 1:25,000): Abeer W Saeed, Alfredo Ribeiro-da-Silva. Non-peptidergic primary afferents are presynaptic to neurokinin-1 receptor immunoreactive lamina I projection neurons in rat spinal cord. Molecular Pain 2012, 8:64 doi:10.1186/1744-8069-8-64.

Images: CGRP, IB4 and P2X3 staining in transverse spinal cord sections. A and B show low magnification confocal images of CGRP-IR and IB4 positive (A) or P2X3-IR (B) fibers. C and D represent high magnification confocal images from the middle third of the lateromedial extent of the superficial dorsal horn. In C, note that there is limited co-localization of IB4 and CGRP (in yellow). Arrowheads show axonal varicosities (boutons) from nonpeptidergic fibers in lamina I, which do not co-localize CGRP immunoreactivity. The framed regions in A and B indicate the approximate regions from where C and D, respectively, were obtained (the latter originate from other sections). CGRP (in green); IB4 (in red); P2X3 (in red). Scale bar (A, B) = 200 μm; scale bar (C, D) = 20 μm

Tissue processing: The injection site at the level of the parabrachial nucleus was examined by cutting serial, 100 μm-thick coronal sections of the relevant brain region. The dorsal aspect of the L4-L5 spinal cord segment was cut into serial, 50 μm-thick horizontal sections (n = 10), 50 μm-thick parasagittal sections (n = 4) or 50 μm-thick transverse sections (n = 4). All sections were cut using a freezing sledge microtome (Leica, Richmond Hill, Ontario) and collected as freefloating in phosphate-buffered saline (PBS) with 0.2% Triton-X 100 (PBS + T). To block unspecific staining, all spinal cord sections were incubated, for one hour, in 10% normal donkey serum (NDS) (Jackson, West Grove, PA) in PBS + T at room temperature. Subsequently, the sections were placed in primary antibodies (or conjugated lectin IB4 - see below) for 48 hours at 4 °C. We used a mixture of 2 or 4 primary antibodies (each raised in a different species), or IB4, in PBS + T containing 5% NDS. Next, the sections were washed in PBS + T and then incubated in species-specific secondary antibodies that were raised in donkey and conjugated to either AlexaFluor 488, AlexaFluor 405, Rhodamine RedX or biotin. The sections were incubated in 3 different cocktails: #1) rabbit anti-CGRP at a 1:200 dilution (Sigma, St Louis, MO) and lectin IB4 conjugated to AlexaFluor 568 at a 1:200 dilution (Molecular Probes); #2) rabbit anti-CGRP and guinea pig anti-P2X3 at a 1:25,000 dilution (Neuromics, Edina, MN); #3) goat anti-CTb at a 1:5000 dilution (List Biological), rabbit anti-NK-1r at a 1:10000 dilution (Sigma, St Louis, MO), guinea pig anti-CGRP at a 1:8000 dilution (Peninsula, San Carlos, CA) and lectin IB4 conjugated to AlexaFluor 647 at a 1:200 dilution (Molecular Probes). All the sections were washed with PBS + T and then (for #1) incubated for 2 hours at room temperature with donkey anti-rabbit AlexaFluor 488; (for #2) incubated for 90 minutes in a biotin conjugated donkey anti-guinea pig IgG (Jackson Immunoresearch, West Grove, PA, 1:200). Further signal amplification was achieved by treating the sections with 1 hour incubation in an avidin-biotin (A + B) complex (Vectastain Elite ABC kit, Vector Laboratories) followed by tyramide (Perkin-Elmer, Norwalk, CT, 1:75) for 7 minutes. Sections were then incubated in streptavidin conjugated to AlexaFluor 568 (Molecular Probes, Eugene, OR, 1:200) and donkey anti-rabbit AlexaFluor 488; or (for #3) incubated for 2 hours at room temperature with secondary antibodies: donkey anti-goat Rhodamine Red X, donkey anti-rabbit AlexaFluor 488, and donkey anti-guinea pig AlexaFluor 405. Finally, sections were washed with PBS, mounted on gelatin-subbed slides and coverslipped with an anti-fading mounting medium (Aqua Polymount; Polysciences, Warrington, PA). Slides were stored at −4 °C pending further processing.

I will continue to publish outstanding customer data/images using our natibodies/markers.

Sunday, October 14, 2012

Teminally Differentiated Human Chondrocytes

Save 20% on New Chondrocytes through November 30, 2012

Certain customers tell me they purchase our UCB and eSC Derived Human Mesenchymal Stem Cells to grow and differentiate into Chondrocyte cultures for the study of joint disease.

Our goal is to save time and expense in the development of your cell based assays. We now offer these options:
NameCatalog #TypeSpeciesApplicationsSizePrice
Native Human Chondrocytes-PilotSC00A7-100KPrimary CellsHCell Assays100,000 Cells$245
Native Human Chondrocytes-HCSSC00A7-500KPrimary CellsHCell Assays500,000 Cells$449
Native Human Chondrocytes-HTS PilotSC00A7-4000KPrimary CellsHCell Assays4X1,000,000 Cells$2,399
Native Human Chondrocytes-HTSSC00A7-1000KPrimary CellsHCell Assays1,000,000 Cells$799
Fluorescein Labeled Human Chondrocytes-PilotSC00A8-100KPrimary CellsHCell Assays100,000 Cells$295
Fluorescein Labeled Human Chondrocytes-HCSSC00A8-500KPrimary CellsHCell Assays500,000 Cells$449
Fluorescein Labeled Human Chondrocytes-HTS PilotSC00A7-1000KPrimary CellsHCell Assays1,000,000 Cells$799
Fluorescein Labeled Human Chondrocytes-HTSSC00A7-4000KPrimary CellsHCell Assays4X1,000,000 Cells$2,399
Rhodamine Labeled Human Chondrocytes-PilotSC00A9-100KPrimary CellsHCell Assays100,000 Cells$295
Rhodamine Human Chondrocytes-HCSSC00A9-500KPrimary CellsHCell Assays500,000 Cells$449
Rhodamine Labeled Human Chondrocytes-HTS PilotSC00A9-1000KPrimary CellsHCell Assays1,000,000 Cells$799
Rhodamine Labeled Human Chondrocytes-HTSSC00A9-4000KPrimary CellsHCell Assays4X1,000,000 Cells$2,399
Human Chondrocytes MediaSC00PC3-100Cell Growth MediaH100 ml
500 ml

Images: Chondrocyte cultures.

We plan on continuing to add new potent and pure primary cells to accelerate meaning results from basic disease research and drug discovery.

Wednesday, October 03, 2012

Neuropeptide Ys-Regulate Food Intake & Body Weight

Role of salivary PYY in the modulation of food intake.

This is a study that comprehensively shows the expression of Neuropeptide Ys  (PYY 3–36 ) are present in saliva and showed the expression of its preferred receptor, Y2R, in the basal layer of the progenitor cells of the tongue epithelia and von Ebner's gland. The researchers used our Y2R antibody (dilution 1:3000) to determine level of expression. This receptor appears to be the main player in mediating hunger and body weight: Hurtado MD, Acosta A, Riveros PP, Baum BJ, Ukhanov K, et al. (2012) Distribution of Y-Receptors in Murine Lingual Epithelia. PLoS ONE 7(9): e46358. doi:10.1371/journal.pone.0046358.

Images: Immunolocalization of Y1, Y2, Y4, and Y5 receptors (Rs) in the dorsal epithelium of murine tongue. Images: Mirror section pairs (Panels A and B, C and D, E and F) were hybridized to the respective YR antibody (green), followed by DAPI counterstain (blue), as indicated in the upper left corner of each panel. For better viewing, the confocal images in B, D, and F were reflected horizontally. Representative areas of the epithelium, positive for either YR (dashed rectangles in the left-sided panels), are shown as close-up images on the right next to each respective panel. The irregular columned structures at the epithelial surface are transversely sectioned filiform papillae. G - Y4R-positive neuronal fibers (green) are located in the subepithelial region underlying the basal laminae. H – co-localization of Y4R and NCAM (red) immunoreactivity within mechanoreceptors of Meissner corpuscles (MC). As a morpho-histological reference of the dorsal lingual epithelium structure, an hematoxylin and eosin stained section is shown in panel I. Panel J shows a hypothetical diagram of a lingual dorsal epithelium layer and the differentiation/migration lineage of cell types expressing respective YR subtypes. K5 – cytokeratin-5 [2]. doi:10.1371/journal.pone.0046358.g006.

Note: this study is further validation of the potency of our NPY Y2 antibody.

Conclusion: NPY family peptides and their cognate receptors in the oral cavity may mediate a wide variety of functions, including proliferation, differentiation, motility, taste perception, as well as satiation. All of these multiple functions and their respective molecular mechanisms are subjects of the ongoing investigations.

Understanding these molecular mechanisms could provide the foundation for discovering less intrusive therapies for obesity. I will keep you posted.