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: Hippocampus

Article Title: Septohippocampal pathways contribute to system consolidation of a spatial memory: sequential implication of GABAergic and cholinergic neurons.

doi: 10.1002/hipo.20837

Figure Lengend Snippet: FIGURE 4. Representative images of ChAT (top) and Parv (bottom) immunostainings in SHAM (A and E), SAP (B and F), OREX (C and G), and SAP/OREX (D and G) rats. For anti-Parv immunostaining, a slice showing nonspecific staining (no primary antibody used during incubation) is also shown. In the MS/vDBB regions (plate modified from Paxinos and Watson, 1998), the two squares on each side delimit the regions where ODs for anti-Parv immunostaning were measured. Cholinergic neurons were mark- edly depleted in SAP (C) and SAP/OREX (D) and relatively spared in OREX (B) rats. GABA neurons were markedly depleted in OREX (G) and SAP/OREX (H) and relatively preserved in SAP (F) rats. Both neuronal populations were unaltered in SHAM animals (A and E).

Article Snippet: As primary antibodies, we used a goat polyclonal antibody directed against ChAT (1:500; Chemicon International, Temecula, CA) and a mouse monoclonal antibody directed against Parv (1:4000; Sigma-Aldrich, P 3088, St. Louis, MO), whereas donkey anti-goat (ChAT) and horse anti-mouse (Parv) biotinylated secondary antibody (1:500, Vector Laboratories International, Burlingame, CA) were used.

Techniques: Immunostaining, Staining, Incubation

Journal: Scientific Reports

Article Title: Catalytically active tissue transglutaminase colocalises with Aβ pathology in Alzheimer’s disease mouse models

doi: 10.1038/srep20569

Figure Lengend Snippet: Serial sagittal whole brain sections of wild-type and APP23 mice were incubated with the TG substrate BAP or the anti-Aβ antibody and visualised using the DAB chromogen. The anti-Aβ antibody demonstrated Aβ plaques in 27-months old mice ( b ). BAP staining was found in the cerebral blood vessel walls of both 7-months old wild-type ( a ) and 27-months old APP23 mice ( c ). In addition, in APP23 mice BAP staining was present in Aβ plaque-like structures ( c ). Double immunofluorescence using the anti-Aβ antibody and BAP staining confirmed the presence of TG activity in Aβ plaques in 12-months old mice ( d–f ) as well as in cerebral vessel walls ( e , arrow). BAP staining was found in the majority of dense core plaques, although it was absent from the cores of these plaques, confirmed by double immunofluorescence of ThioS with BAP ( g–i ). Co-incubation of BAP with the selective tTG inhibitor Z-DON (100 μM) blocked the tTG-catalysed incorporation of BAP ( j–l ). Scale bars: ( c , d ) 36 μm, ( a , d – f , j –l) 30 μm, ( g – i ) 15 μm. Abbreviations: Aβ = amyloid beta, BAP = biotinylated 5-(biotinamido)-pentylamine, ThioS = Thioflavin S, tTG = tissue transglutaminase, Z-DON = Z-DON-Val-Pro-Leu-OMe.

Article Snippet: The secondary antibodies, biotinylated goat anti-rabbit or donkey anti-goat (dilution 1:400; Jackson Immunoresearch Laboratories Inc., Suffolk, UK) were diluted in 3% BSA/TBS-T and incubated for 2 hours at room temperature followed by incubation with the avidin-biotin complex (ABC, 1:400 in TBS/T; Vector Laboratories Inc., Burlingame, CA, USA) for 1 hour.

Techniques: Incubation, Staining, Immunofluorescence, Activity Assay

Journal: Scientific Reports

Article Title: Catalytically active tissue transglutaminase colocalises with Aβ pathology in Alzheimer’s disease mouse models

doi: 10.1038/srep20569

Figure Lengend Snippet: Double immunofluorescence was performed with an anti-Aβ antibody or ThioS with BAP or T26 resulting in double stainings Aβ/BAP, Aβ/T26, ThioS/BAP and ThioS/T26. Only well-defined anti-Aβ antibody positive or ThioS-positive plaques were counted. Well-defined plaques are marked with arrows, both for Aβ ( a ) and ThioS ( b ) staining (APP23 staining shown), whereas examples of plaques that were not taken into quantification are marked with asterisks ( a,b ). The percentages of anti-Aβ antibody positive and ThioS positive plaques positive for BAP or T26 are shown for both APP23 and APP/PS1 mice ( c ). Non-parametric Kruskal-Wallis testing demonstrated a significant higher percentage of ThioS positive plaques with BAP (77.3 ± 1.9% Mean ± SEM) or T26 (73.4 ± 5.2%) staining in APP23 mice compared to APP/PS1 mice where BAP and T26 staining were present in 50.5 ± 8.0% or 38.3 ± 8.1% of the ThioS positive plaques respectively (p = 0.02). In APP23, a trend increase of the percentage of Aβ plaques with T26 staining was present compared to APP/PS1 mice (66.1 ± 6.9% versus 35.5 ± 8.0% respectively, p = 0.06). The increased percentage of Aβ plaques with BAP staining in APP23 mice (61.0 ± 6.9%) compared to APP/PS1 mice (45.7 ± 7.8%) did not reach statistical significance . Error bar: 300 μm. Abbreviations: Aβ = amyloid beta, BAP = biotinylated 5-(biotinamido)-pentylamine ThioS = Thioflavin S, tTG = tissue transglutaminase. *p < 0.05, # p < 0.1. Mean ± SEM is displayed.

Article Snippet: The secondary antibodies, biotinylated goat anti-rabbit or donkey anti-goat (dilution 1:400; Jackson Immunoresearch Laboratories Inc., Suffolk, UK) were diluted in 3% BSA/TBS-T and incubated for 2 hours at room temperature followed by incubation with the avidin-biotin complex (ABC, 1:400 in TBS/T; Vector Laboratories Inc., Burlingame, CA, USA) for 1 hour.

Techniques: Immunofluorescence, Staining