Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: N-glycosylation of the prolactin receptor is not required for activation of gene transcription but is crucial for its cell surface targeting.

doi: 10.1210/mend.12.4.0085

Figure Lengend Snippet: Fig. 4. Perinuclear Localization of the Aglycosylated PRLR COS-7 cells transfected with WT (a and d), N80,108D (b and e), or N35,80,108D (c and f) PRLR cDNAs were fixed in 4% paraformaldehyde (a, b, and c: nonpermeabilized conditions) or in methanol, 220 C (d, e, and f: permeabilized conditions). Mouse monoclonal antibody U5 (160 mg/ml) followed by FITC goat anti-IgG (dilution 1:40) were used, as described in Materials and Methods. Note the immunostaining in the juxtanuclear area of the aglycosylated PRLR in permeabilized cells (f) and the absence of expression at the plasma membrane level in nonpermeabilized cells (c). 293 cells transfected with WT (g), N80,108D (h), or N35,80,108D (i) PRLR cDNAs were processed for fixation (4% paraformaldehyde) and immunofluorescence staining, as described for COS-7 cells. Note the absence of immunostaining at the cell surface and low perinuclear staining of the aglycosylated receptor in nonpermeabilized conditions (i). Magnification, 3400.

Article Snippet: The secondary antibodies used in these studies were the Texas-Red-conjugated and fluorescein-isothiocyanate-conjugated (FITC) goat anti-mouse IgG (dilution 1:40) (Vector Laboratories, Burlingame, CA; and Biosys, Compiègne, France), the biotinylated donkey anti-rabbit IgG and the Texas-Red-conjugated streptavidin (1:100; Amersham International, Aylesbury, UK).

Techniques: Transfection, Immunostaining, Expressing, Clinical Proteomics, Membrane, Immunofluorescence, Staining

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: N-glycosylation of the prolactin receptor is not required for activation of gene transcription but is crucial for its cell surface targeting.

doi: 10.1210/mend.12.4.0085

Figure Lengend Snippet: Fig. 5. Deglycosylated PRLR Colocalizes with the Golgi rab6 in COS-7 Cells a, Labeling of fixed permeabilized COS-7 cells transfected with N35,80,108D receptor using U5 antibody. Panels b and c and panels d and e show a double- labeling experiment of fixed permeabilized COS-7 cells transfected with the N35,80,108D mutant, with anti-PRLR antibody [visualized with a secondary anti-mouse FITC secondary antibody (b and d)], and anti-Golgi antibody [anti-rab6 visualized with bioti- nylated secondary anti-rabbit IgG followed by Texas-Red- conjugated streptavidin (c and e)]. The choice of mouse anti- PRLR and rabbit anti-Golgi IgG as primary antibodies allows accurate double-labeling experiments. Panels b-c and d-e show two different COS-7 cells. f, Micrographs of COS-7 cells stained for anti-protein disulfide isomerase (endoplas- mic reticulum protein) antibody, visualized with Texas-Red- conjugated anti-mouse secondary antibody. Magnification, 3400.

Article Snippet: The secondary antibodies used in these studies were the Texas-Red-conjugated and fluorescein-isothiocyanate-conjugated (FITC) goat anti-mouse IgG (dilution 1:40) (Vector Laboratories, Burlingame, CA; and Biosys, Compiègne, France), the biotinylated donkey anti-rabbit IgG and the Texas-Red-conjugated streptavidin (1:100; Amersham International, Aylesbury, UK).

Techniques: Labeling, Transfection, Mutagenesis, Staining

Journal: Histochemistry and cell biology

Article Title: Stem cell marker TRA-1-60 is expressed in foetal and adult kidney and upregulated in tubulo-interstitial disease

doi: 10.1007/s00418-010-0741-7

Figure Lengend Snippet: TRA-1-60 and Pax-2 immunostaining of normal human kidney at 10 weeks of gestation. a and c show TRA-1-60 expression in the nephrogenic zone. a TRA-1-60 was intensely expressed on the apical surface of the ureteric bud and putative collecting ducts. c TRA-1-60 expression in the ureteric bud tip at higher magnification. b and d show Pax-2 expression in the nephrogenic zone. b Transcription factor Pax-2 was strongly expressed in the ureteric bud, collecting duct and also in condensing metanephric mesenchyme and derivatives including comma-shaped bodies. A lower level of expression was noticeable in maturing nephrons. d Pax-2 expression in the ureteric bud, condensing mesenchyme and S-shaped bodies at higher magnification. e Mouse IgM, isotype control for TRA-1-60 immunostaining. f Rabbit IgG negative control for Pax-2 staining. ub ureteric bud, cd collecting duct, mm metanephric mesenchyme, cb comma-shaped body, Sb S-shaped body, nephr nephron. Scale bars 60 μm

Article Snippet: Control antibodies at similar concentrations to primary antibodies were used: rabbit IgG (Vector Laboratories) for Pax2, mouse IgM for TRA-1-60, mouse IgG1 for Ki 67 and mouse IgG2a for EMA, all from Sigma.

Techniques: Immunostaining, Expressing, Negative Control, Staining

Journal: Histochemistry and cell biology

Article Title: Stem cell marker TRA-1-60 is expressed in foetal and adult kidney and upregulated in tubulo-interstitial disease

doi: 10.1007/s00418-010-0741-7

Figure Lengend Snippet: TRA-1-60 antigen expression in tubulo-interstitial damage. Serial sections of areas of renal cortex with tubulo-interstitial damage were stained for EMA (a, d, g), TRA-1-60 (b, e, h) and LTA (c, f, i). b Shows normal kidney with accumulation of TRA-1-60 expressing cells (arrows) in scarred areas marked by lymphocyte infiltration (arrowheads). The TRA-1-60 expressing tubules were also positive for EMA (a arrows) but not LTA lectin (c arrows) on serial sections. Increased TRA-1-60 expression by tubular cells in the cortex affected by ATN (e arrows) and GN (h arrows). On serial sections the TRA-1-60 expressing tubules were EMA positive (d and g, respectively, arrows) and LTA lectin negative (f and i, respectively, arrows). j–l Show negative controls: mouse IgG2 for EMA antibody (j), mouse IgM for TRA-1-60, with TBS only for LTA lectin. Scale bar 60 μm

Article Snippet: Control antibodies at similar concentrations to primary antibodies were used: rabbit IgG (Vector Laboratories) for Pax2, mouse IgM for TRA-1-60, mouse IgG1 for Ki 67 and mouse IgG2a for EMA, all from Sigma.

Techniques: Expressing, Staining

Journal: Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology

Article Title: Behavioral and neurochemical alterations in mice deficient in anaplastic lymphoma kinase suggest therapeutic potential for psychiatric indications.

doi: 10.1038/sj.npp.1301446

Figure Lengend Snippet: Figure 1 Generation of ALK KO mice. (a) Gene deletion strategy. (b) Genotyping of ALK receptor KO mice. Typical gel electrophoresis example showing homozygous mice with the 351 bp targeted product only, WT mice with the 187 bp endogenous product only, and heterozygous with both products. HO, homozygous; HE, heterozygous; WT, wild-type mice. Figure 2 (a) Quantitative analysis of relative expression levels of ALK transcripts in cerebellum, hippocampus, and frontal cortex in HOs vs WT littermates (n ¼ 6 animals/genotype). Data are normalized to a-tubulin and expressed as relative fold-change. Notice that the relative expression of ALK transcripts (as judged by the N-terminal PCR probe) is higher in the frontal cortex and the cerebellum than in the hippocampus (9.3- and 2.6- fold higher compared with hippocampal levels of this probe). Quantification of ALK products by qPCR in the HO animals shows lack of expression of mRNA transcripts encoding the 30 end of ALK (c-2 and c-3 probes), as expected from the gene disruption strategy. Notice varying amounts of residual 50 transcript across these brain regions. (b) Relative transcript expression of various genes implicated in ALK signalling or neurochemical pathways affected in the HO animals. Data is plotted as relative expression changes in HO vs WT animals in the frontal cortex and hippocampus. With the exception of ALK transcripts, we detected no statistically significant changes in any of the additional genes monitored.

Article Snippet: The sections were then blocked for endogenous mouse IgG with a vector mouseon-mouse kit and for nonspecific antibody binding with 0.1% BSA for 30 min.

Techniques: Nucleic Acid Electrophoresis, Expressing, Disruption

Journal: BMC Molecular Biology

Article Title: Receptor protein tyrosine kinase EphB4 is up-regulated in colon cancer

doi: 10.1186/1471-2199-2-15

Figure Lengend Snippet: Immunohistochemical localisation of EphB4 expression in three different colon cancers and matched normal mucosa. The brown stain from the biotinylated secondary antibody indicates the EphB4 protein. Nuclei are stained with Harris haematoxylin and appear blue. High power (100X) magnification images of three different adenocarcinomas (well differentiated, moderately well differentiated and poorly differentiated) and their matched normal mucosa are shown. Strong staining of the tumour tissue and very weak, diffuse staining of normal tissue was evident for each sample set. There was no cross-reactivity with the secondary antibody alone (result not shown).

Article Snippet: After rinsing with PBS, the sections were incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories) for 30 min at room temperature followed by washing with PBS.

Techniques: Immunohistochemical staining, Expressing, Staining

Journal: Journal of Neuroinflammation

Article Title: Interleukin-1α expression precedes IL-1β after ischemic brain injury and is localised to areas of focal neuronal loss and penumbral tissues

doi: 10.1186/1742-2094-8-186

Figure Lengend Snippet: IL-1α is expressed by microglia localized to focal neuronal and BBB injury 24 h after MCAo . Images are coronal sections from brains of C57BL6/H and CX3CR1-GFP +/- mice 60 min MCAo and 24 h reperfusion. Widefield images show IL-1α-expressing (red), GFP positive (green) microglia in ipsilateral (Ai), not contralateral (ii) amygdala 24 h after MCAo in a CX3CR1-GFP +/- mouse. IL-1α immunohistochemistry with cresyl violet co-staining localises IL-1α expressing microglia to the peri-infarct zone in thalamus (Bi) and cortex (Bii) of a C57BL6/H mouse. Focal IgG staining (red) co-localized with IL-1α positive microglia (green) in the ipsilateral cortex of a C57BL6/H mouse (Ci). No IgG or IL-1α staining detected in the contralateral cortex (Cii). IL-1α positive microglia detected in larger areas of IgG staining in the ipsilateral (Ciii), but not contralateral (Civ) hemisphere. Co-localization of IL-1α positive microglia (red) with areas of neuronal loss (blue) in a C57BL6/H mouse (D). Occasional IL-1α positive microglia also found in areas where neurons were morphologically intact (D, inset). Confocal images (E) are maximum Z projections (Ei, iii) and confocal slices at the level of the nucleus (Eii, iv) of IL-1α expressing, GFP positive microglia in a CX3CR1-GFP +/- mouse. Cells with (Ei, ii), and without (Eiii, iv) nuclear IL-1α. Nuclear fluorescence intensities for IL-1α and GFP were quantified from confocal images, and the fold enrichment of IL-1α and GFP in microglial nuclei was calculated in comparison to whole cell fluorescence (F). All images are representative of n ≥ 3 mice. Quantification is of n = 4 CX3CR1-GFP +/- mouse brains, with each data point representing an individual cell, n ≥ 30 cells per brain.

Article Snippet: Staining of coronal brain sections from C57BL6/H mice (Figure ) and CX3CR1-GFP+/- mice (not shown) 24 h after MCAo for IL-1α and IgG (BA-2000, Vector Labs, biotinylated horse anti-mouse IgG, 2 μg/mL; S-32356, Invitrogen, Alexa 594 conjugated streptavidin, 5 μg/mL) revealed that IL-1α expressing cells co-localized to areas of focal BBB damage, mainly near the penumbral regions of the ipsilateral hemisphere (Figure ).

Techniques: Expressing, Immunohistochemistry, Staining, Fluorescence