Journal: International Journal of Molecular Sciences

Article Title: A2A-D2 Heteromers on Striatal Astrocytes: Biochemical and Biophysical Evidence

doi: 10.3390/ijms20102457

Figure Lengend Snippet: A2A-D2 heterodimers on rat striatal astrocytes: proximity ligation assay. Detection of in situ PLA A2A-D2 heteroreceptor complexes was carried out with primary antibodies (rabbit polyclonal anti-A2AR, mouse monoclonal anti-D2R and goat polyclonal anti-GFAP) in rat striatal slices. ( A ) The merge of the maximum intensity projections of a representative field (240x240 µm; z 10 µm) is shown; GFAP (red), DAPI (blue), PLA for A2A-D2 heteroreceptor complexes appears as yellow clusters. The boxed region (i) is shown at a higher magnification in ( B ). ( C ) Merged confocal images of a single z stack of the boxed region (ii) at a higher magnification: double immunolabeling for GFAP (red) and PLA A2A-D2 heteroreceptor complexes (green). ( D ) Colocalized map of the boxed region (ii); the colocalization analysis plugins—colocalization threshold with GFAP as ROI—was used to create the colocalized map; ImageJ Fiji software. A complete lack of stain for PLA A2A-D2 heteroreceptor complexes was obtained in the negative control experiments, performed avoiding the conjugation of a primary antibody with the Duolink Probes. In the figure the merges of the maximum intensity projections of two representative fields are shown: PLA for A2A-D2. heteroreceptor complexes without the primary anti-A2A antibody ( E ) or the primary anti-D2 antibody ( F ). Scale bar 25 µm or 10 µm

Article Snippet: In situ PLA was performed on 10μm rat striatal slices using the primary antibodies (mouse anti-A2A (1:200, Merck Millipore Corporation, 05-717, Burlington, MA, USA); rabbit anti-D2R (1:200, Alomone Labs, ADR-002, Jerusalem, Israel); goat anti-GFAP (1:500, Santa Cruz Biotechnoloy Inc, Dallas, TX, USA, sc-6170), the Duolink in situ PLA detection kit (DUO92014, Sigma-Aldrich, St Louis, MO, USA) and Alexa Fluor 546-conjugated donkey anti-goat (1:500; Molecular Probes, Eugene, OR, USA).

Techniques: Proximity Ligation Assay, In Situ, Confocal, Immunolabeling, Analysis, Software, Staining, Negative Control, Conjugation Assay

Journal: Reproductive biomedicine online

Article Title: Social psychogenic stress promotes the development of endometriosis in mouse.

doi: 10.1016/j.rbmo.2016.11.012

Figure Lengend Snippet: Figure 4 – Immmunoreactivity staining of different markers in ectopic lesions in different groups. (A) Representative immunostaining of ADRB2 in endometrium in CONTROL and SHAM mice and in ectopic endometrium in STRESSED and UNSTRESSED mice. ADRB2 and DRD2 immunoreactivity was both seen primarily in glandular epithelial cells and was localized in the cytoplasm. Scale bar = 125 μm. (B) Representative immunostaining of DRD2, VEGF, CD31, CD41, F4/80, PCNA and α-SMA in the ectopic lesions in UNSTRESSED and STRESSED groups. VEGF immunoreactivity was seen primarily in glandular epithelial cells and was localized in the cytoplasm. CD31 immunostainings were seen mostly in vascular endothelial cells. CD41 shows the extent of platelet aggregation and F4/80 immunoreactivity represents the extent of macrophage infiltration. PCNA immunoreactivity was seen both in glandular epithelial cells and stromal cells were localized in the cell nucleus, but the change of immunoreactivity in glandular epithelial cells was more obvious. α-SMA staining were seen mostly in the stromal component of the ectopic lesions. Scale bar = 125 μm. Mice in stressed (STRS) and unstressed (UNSTRS) groups had undergone endometriosis-inducing surgery, while mice the SHAM group underwent non-endometriosis-inducing surgery. Mice in unstressed (UNSTRS), SHAM and control (CTL) groups were not exposed to stress.

Article Snippet: For negative controls, the immunoglobulin G (IgG) from the rabbit serum (Sigma, Darmstadt, Germany) was used instead of primary antibodies against ADRB2, DRD2, VEGF, CD31, CD41, α-SMA and PCNA except for F4/80, for which the IgG was from the rat serum (Boster, Wuhan, China) was used, all used at the same concentration with its corresponding primary antibody.

Techniques: Staining, Immunostaining, Control