rabbit anti otr  (Danaher Inc)

Journal: International Journal of Molecular Sciences

Article Title: Control of Dopamine Signal in High-Order Receptor Complex on Striatal Astrocytes

doi: 10.3390/ijms25168610

Figure Lengend Snippet: Striatal astrocytes express the A2A, D2, and OT receptors. Representative confocal images showing the presence of the A2A, D2, and OTR in rat striatum and their co-localization with GFAP. Immunofluorescence analysis was conducted in rat hemibrain slices (see for details). Maximum intensity projections and their merges for representative fields (with a dimension of 60 × 60 µm; at least 11 z-stacks) are shown; GFAP (green, ( A , C , E , G , H , J )), A2A (red, ( B , C )), D2 (red, ( F , G )), and OTR (red, ( I , J )). Astrocytes were positively stained with mouse anti-GFAP (red ( K , M )) and goat anti-GFAP (blue ( L , M )) primary antibodies. The scale bars are indicated in the merged images. ( D ) Cresyl violet staining of a close-up section used in . GFAP, glial fibrillary acidic protein; A2A, adenosine receptor; D2, dopamine receptor; OTR, oxytocin receptor.

Article Snippet: The primary antibodies (rabbit anti-OTR, rabbit anti-A2A receptor, and rabbit anti-D2 receptor) were purchased from Alamone Labs (Jerusalem, Israel), while mouse anti-flotillin-1 from BD Biosciences was purchased from DBA Italia (Segrate, Italy).

Techniques: Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Control of Dopamine Signal in High-Order Receptor Complex on Striatal Astrocytes

doi: 10.3390/ijms25168610

Figure Lengend Snippet: Astrocytes express the A2A receptors and D2-OT heteromers. Representative confocal images showing the presence of the D2-OTR heteromers in rat striatum and their co-localization with A2A receptors. The immunofluorescence analysis was conducted by combing the in situ PLA with anti-D2 receptor and anti-OTR primary antibodies, and the classical immunofluorescence technique using anti-A2A receptor, anti-GFAP, and anti-ezrin primary antibodies in rat hemibrain slices (for further details, see Materials and Methods). ( A – E ) Maximum intensity projections for single channels and the merged images of a representative field (29.13 × 29.13 µm; 11 z stacks) are shown; GFAP + ezrin (blue, ( A , E )), PLA D2-OTR (green, ( B , D , E )), and A2A (red, ( C – E )). ( F – J ) Enlarged images for each channel and the merged confocal images of a single z-stack is shown; this is the same field of the maximum intensity projections shown in ( A – E ); GFAP + ezrin (blue, ( F , J )), PLA D2-OTR (green, ( G , I , J )), and A2A (red, ( H – J )). The yellow line in ( J ) was used to create the profile plot shown in ( K ). The colours of the different lines (blue for the GFAP + ezrin signal, green for the PLAD2-OTR signal, red for the A2A signal) correspond to those used in image ( J ). ( L ) A representative merge control image obtained without the primary antibody for OTR or D2 during the PLA protocol. GFAP, glial fibrillary acidic protein; A2A, adenosine receptor; D2, dopamine receptor; OTR, oxytocin receptor; PLA, proximity ligation assay.

Article Snippet: The primary antibodies (rabbit anti-OTR, rabbit anti-A2A receptor, and rabbit anti-D2 receptor) were purchased from Alamone Labs (Jerusalem, Israel), while mouse anti-flotillin-1 from BD Biosciences was purchased from DBA Italia (Segrate, Italy).

Techniques: Immunofluorescence, In Situ, Control, Proximity Ligation Assay

Journal: International Journal of Molecular Sciences

Article Title: Control of Dopamine Signal in High-Order Receptor Complex on Striatal Astrocytes

doi: 10.3390/ijms25168610

Figure Lengend Snippet: Astrocytic processes express A2A, D2, and OTRs. Representative confocal images showing the presence of the studied receptors in rat striatal astrocytic processes and their co-localization with GFAP. The immunofluorescence analysis was conducted using anti-D2 receptor, anti-OTR, anti-A2A receptor, and anti-GFAP primary antibodies in gliosomes (for further details, see Materials and Methods). ( A – E ) A representative field is shown; A2A (green, ( A , E )), D2 (red, ( B , E )), OTR (blue, ( C , E )), and GFAP (yellow, ( D , E )). GFAP, glial fibrillary acidic protein; A2A, adenosine A2A receptor; D2, dopamine D2 receptor; OTR, oxytocin receptor.

Article Snippet: The primary antibodies (rabbit anti-OTR, rabbit anti-A2A receptor, and rabbit anti-D2 receptor) were purchased from Alamone Labs (Jerusalem, Israel), while mouse anti-flotillin-1 from BD Biosciences was purchased from DBA Italia (Segrate, Italy).

Techniques: Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Control of Dopamine Signal in High-Order Receptor Complex on Striatal Astrocytes

doi: 10.3390/ijms25168610

Figure Lengend Snippet: Co-immunoprecipitation of the OTR, A2A, and D2 receptors in striatal astrocytic processes. The aliquots (300 µg) of Triton X-100-soluble proteins prepared from striatal fresh isolated gliosomes were immunoprecipitated with 1 µg of anti-OTR ( A ), or anti-A2A ( B ), or anti-D2 ( C ) antibody. The immunoprecipitated (IP) and not immunoprecipitated (O, output) materials were analyzed by Western blot (WB) for the indicated antigens. A2A, OTR, D2, and flotillin-1 (FLOT) immunoreactive bands were quantified and the data are reported in the graphs as a percentage of the total amount of the relevant protein (% of total). The values are means ± SEM ( n = 5 for ( A , C ); n = 4 for ( B )). For each protein, a representative blot is shown. The arrows indicate the expected weights of the antigens. A2A, adenosine A2A receptor; D2, dopamine D2 receptor; OTR, oxytocin receptor; FLOT, flotillin.

Article Snippet: The primary antibodies (rabbit anti-OTR, rabbit anti-A2A receptor, and rabbit anti-D2 receptor) were purchased from Alamone Labs (Jerusalem, Israel), while mouse anti-flotillin-1 from BD Biosciences was purchased from DBA Italia (Segrate, Italy).

Techniques: Immunoprecipitation, Isolation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Control of Dopamine Signal in High-Order Receptor Complex on Striatal Astrocytes

doi: 10.3390/ijms25168610

Figure Lengend Snippet: Endogenous glutamate efflux in response to 4-AP-induced depolarization in striatal gliosomes. Modulation by the D2-OTR heteromers and A2A receptors. The inhibitory effect of OT 3 nM and quinpirole 0.1 µM on the 4-AP-evoked endogenous glutamate release; abolishment by CGS 21,680 0.01 µM and the antagonism of the A2A receptors by SCH-58261. The bars represent the overflow of the glutamate release, expressed as pmol/mg of protein, in the presence of drugs at the concentrations used. Briefly, 4-AP was added (6 min) during superfusion; OT, quinpirole, and CGS 21,680 were added together with 4-AP, while SCH-58261 was added 8 min before the agonists. Further experimental details can be found in Materials and Methods. Data are the mean ± SEM of n = 5–10 independent experiments. * p < 0.0001 compared with the effect of 4-AP; # p < 0.0001 compared with the effect of 4-AP + OT + quinpirole; $ p < 0.0001 compared with the effect of 4P + OT + quinpirole + CGS 21,680 by one-way ANOVA plus Tukey’s post hoc test. 4-AP, 4-aminopyridine; OT, oxytocin.

Article Snippet: The primary antibodies (rabbit anti-OTR, rabbit anti-A2A receptor, and rabbit anti-D2 receptor) were purchased from Alamone Labs (Jerusalem, Israel), while mouse anti-flotillin-1 from BD Biosciences was purchased from DBA Italia (Segrate, Italy).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Control of Dopamine Signal in High-Order Receptor Complex on Striatal Astrocytes

doi: 10.3390/ijms25168610

Figure Lengend Snippet: Predicted models of the A2A-D2-OTR heterotrimer. The predicted structure of the receptor complex in an “open” topological arrangement is shown in ( A ). It was derived by first docking A2A and D2 receptors and then associating the OTR. The left panel shows the complex in the membrane environment used for refinement by energy minimization and the resulting heterotrimer is shown in the middle panel. A solution characterized by a “closed” topological arrangement is shown in ( B ). It was obtained by docking the A2A receptor to the previously estimated D2-OTR heterodimer. For both cases, the obtained arrangement of the transmembrane domains (as different coloured numbers for each receptor in the illustration) is illustrated in the right panels together with the estimated change in free energy associated with complex formation. For each topology, the structure shown is the one exhibiting the best energy score according to the applied docking method.

Article Snippet: The primary antibodies (rabbit anti-OTR, rabbit anti-A2A receptor, and rabbit anti-D2 receptor) were purchased from Alamone Labs (Jerusalem, Israel), while mouse anti-flotillin-1 from BD Biosciences was purchased from DBA Italia (Segrate, Italy).

Techniques: Derivative Assay, Membrane

Journal: International Journal of Molecular Sciences

Article Title: Control of Dopamine Signal in High-Order Receptor Complex on Striatal Astrocytes

doi: 10.3390/ijms25168610

Figure Lengend Snippet: Pharmacological tools and their concentration used to modulate the endogenous glutamate efflux or the cytosolic [Ca 2+ ].

Article Snippet: The primary antibodies (rabbit anti-OTR, rabbit anti-A2A receptor, and rabbit anti-D2 receptor) were purchased from Alamone Labs (Jerusalem, Israel), while mouse anti-flotillin-1 from BD Biosciences was purchased from DBA Italia (Segrate, Italy).

Techniques: Concentration Assay