Journal: PLoS ONE

Article Title: Synergy of Combined tPA-Edaravone Therapy in Experimental Thrombotic Stroke

doi: 10.1371/journal.pone.0098807

Figure Lengend Snippet: ( A–L ) Immunostaining performed at 1 h after tCCAo, tHI or permanent carotid ligation (pCCAo) plus hypoxia showed deposits of fibrin(ogen) and platelets (detected by anti-Integrin α2β/CD41) and P-Selectin-positive blood vessels (a marker for endothelial activation) and in the ipsilateral hemisphere following tHI insults (C, G, K). Permanent carotid ligation plus hypoxia produced the same staining pattern, but with stronger signals of all three markers (D, H, L). This staining pattern was absent in the brain after tCCAo (A, E, I) or on the contralateral hemisphere following tHI (B, F, J) (n>4 for each). ( M ) Plasminogen-zymogram showed definitive induction of the brain uPA and ∼50% increase of tPA activity in the ipsilateral hemisphere (R) at 4 h post-HI in neonates. No uPA-induction was found in adult brains at 4 h following tCCAo or tHI insults. The tPA activity in the ipsilateral hemisphere (R) following tHI was reduced to ∼67% of the level in unchallenged (UN) brains (p<0.05). The right panel is the quantification of zymogram band intensity (n = 4 for each). ( N ) The plasminogen-zymogram from plasma showed the lack of tPA activity and a similar degree of uPA activity at 4 h after tCCAo or tHI insults in adult mice. Immunoblot detection of transferrin (Tf) served as the internal control. ( O ) Direct measurement of tPA activity using a fluorescent substrate kit also showed significant reduction in the ipsilateral hemisphere at 4 h following tHI insult in adult brains (n = 3-4 for each). ( P–Q ) Immunoblot analysis showed minimal change of the tPA protein level and no sign of plasminogen activator inhibitor-1 (PAI-1) up-regulation after tCCAo or tHI insult in adult brains (n = 4 for each). Scale bar: 200 μm in A–L.

Article Snippet: The following antibodies were used for immunoblot: rabbit anti-tPA (Molecular Innovations, Novi, MI), mouse anti-PAI-1 (Molecular Innovations), and mouse anti- β-actin (Sigma, St. Louis, MO).

Techniques: Immunostaining, Ligation, Marker, Activation Assay, Produced, Staining, Activity Assay, Western Blot

Journal: Frontiers in Neurology

Article Title: Morphofunctional Organization of the Connections From the Medial and Intermediate Parts of the Central Nucleus of the Amygdala Into Distinct Divisions of the Lateral Hypothalamic Area in the Rat

doi: 10.3389/fneur.2018.00688

Figure Lengend Snippet: Photomicrographs illustrating the co-distribution of PHAL axons, MCH and Hcrt cell bodies using a triple immunofluorescence procedure. (A–C) are low magnification pictures on sections at levels 27 (in fact better seen as inter-level 27/28), 28 and 30. Note the innervation of regions containing MCH and Hcrt cell bodies and corresponding to the dorsal and suprafornical regions of the LHA. However, the ZI, or the caudal perifornical regions (level 30) that are rich in MCH cell bodies but poor in Hcrt neurons are not innervated. By contrast at level 30, the PST that contains a dense group of MCH neurons is intensely innervated. (D,E) Higher magnification illustrations showing that buttons by PHAL axons could be seen close to MCH and Hcrt cell bodies. However, it seemed to be mostly en passant putative contacts.

Article Snippet: After rinsing in PBS-T, free-floating sections were incubated with primary antibodies [anti-Hcrt (Mouse monoclonal, 1:1,000, ANGIO-PROTEOMIE®); anti-MCH [Rabbit polyclonal, 1:1,000, our laboratory , RRID:AB_2616562 ]; anti-PHAL (Goat polyclonal, 1:1,000, Vector Laboratories®, RRID:AB_10000080 )] dissolved in PBS-T, 1% bovine serum albumin, 10% lactoproteins, and 0.01% sodium azide or only in PBS-T for 24 h at 4°C.

Techniques: Immunofluorescence

Journal: Frontiers in Neurology

Article Title: Morphofunctional Organization of the Connections From the Medial and Intermediate Parts of the Central Nucleus of the Amygdala Into Distinct Divisions of the Lateral Hypothalamic Area in the Rat

doi: 10.3389/fneur.2018.00688

Figure Lengend Snippet: Photomicrographs after quadruple labeling experiments with the detection using immunofluorescence of PHAL, MCH, and Hcrt, the fourth signal being the native fluorescence of the FG. Because it is difficult to separate signals from each other on the picture, we added line drawings that schematize the distribution of these signals. Sections are from experiments PHAL#1 (A,B) or PHAL#2 (C,D) respectively, and pass approximately through the level 28. Note that the PHAL distribution is distinct in these two cases, with an innervation restricted to the LHAd in experiment PHAL#1, while the LHAs is innervated in the experiment PHAL#2. Furthermore in this experiment, innervation of FG retrogradely labeled neurons from the PSTN are observed. In fact in a few occasion we could see PHAL axons forming pericellular nets around FG-containing cell bodies lateral to the fornix [framed picture in (C) , optical slice taken using the Apotome feature of the microscope]. FG labeled neurons were observed lateral to the fornix, in an area that belong to the LHAd, and in the far ventrolateral LHA. However, we cannot rule out the possibility that the labeling of these far latero-ventral neurons could not be the result of an uptake of the FG through dendrites extending into the injection site. In the two experiments, as in the experiment AMY1, only passing axons seemed to contact MCH or Hcrt cell bodies.

Article Snippet: After rinsing in PBS-T, free-floating sections were incubated with primary antibodies [anti-Hcrt (Mouse monoclonal, 1:1,000, ANGIO-PROTEOMIE®); anti-MCH [Rabbit polyclonal, 1:1,000, our laboratory , RRID:AB_2616562 ]; anti-PHAL (Goat polyclonal, 1:1,000, Vector Laboratories®, RRID:AB_10000080 )] dissolved in PBS-T, 1% bovine serum albumin, 10% lactoproteins, and 0.01% sodium azide or only in PBS-T for 24 h at 4°C.

Techniques: Labeling, Immunofluorescence, Fluorescence, Microscopy, Injection