Journal: The Journal of Neuroscience

Article Title: Dendritic HCN2 Channels Constrain Glutamate-Driven Excitability in Reticular Thalamic Neurons

doi: 10.1523/JNEUROSCI.1630-07.2007

Figure Lengend Snippet: HCN2 is the major isoform that generates Ih in RTN neurons. A1, Low-magnification confocal microscopy image shows immunofluorescence for parvalbumin (Pv; white) labeling RTN neurons in the thalamus. Scale bars: A1, 200 μm; B1, C1, 5 μm. A2, MAP2 expression (green) is present in parvalbumin-positive GABAergic neurons, as assessed by double immunofluorescence. B1, High-magnification images show triple-immunofluorescent labeling for parvalbumin (blue), MAP2 (green), and HCN2 (red) in a +/+ RTN neuron. Note that very little HCN2-IR is present on the RTN cell body densely labeled with parvalbumin. In the merged image, there is no apparent colocalization between HCN2-IR and MAP2-IR. B2, HCN2 immunolabeling is absent in −/− RTN neurons. C1, Immunostaining with antibodies against HCN4 shows prominent somatic labeling in the +/+ RTN neuron. C2, A similar HCN4 expression pattern is seen in a −/− RTN neuron. Merged images demonstrate that labeling for HCN4 and MAP2 does not overlap in +/+ (C1) and −/− (C2) RTN neurons. D1, D2, Whole-cell voltage-clamp recordings show a family of current traces recorded from a +/+ RTN neuron (D1); the voltage protocol is shown below. HCN2 deletion (D2) eliminates Ih currents in the RTN neuron. E1, E2, Exemplar current traces in response to a 20-s-long voltage step to −110 mV (from −40 mV; protocol not shown) in +/+ (E1) and −/− (E2) RTN neurons. A ZD7288-sensitive current is readily detected in the +/+, but not −/−, RTN neuron.

Article Snippet: Anti-HCN2 and anti-HCN4 rabbit antibodies (1:40 dilution; Alomone Labs, Jerusalem, Israel) were used to detect immunoreactivity (IR) for the corresponding HCN isoforms.

Techniques: Confocal Microscopy, Immunofluorescence, Labeling, Expressing, Immunolabeling, Immunostaining

Journal: Scientific Reports

Article Title: Sonic hedgehog signaling is negatively regulated in reactive astrocytes after forebrain stab injury

doi: 10.1038/s41598-018-37555-x

Figure Lengend Snippet: Gli1 astrocytes marked before injury exhibit features of reactive astrocytes. ( a , f ) Low power micrographs from the contralateral ( a ) and ipsilateral ( f ) cortex of an adult Gli1 CreER /+ ; R26 tdTomato / tdTomato mouse that received tamoxifen 2 weeks before injury. Fluorescence immunohistochemistry for GFAP-S100β (green) shows that Gli1 astrocytes (red) at the lesion site upregulate GFAP and extend long radial processes towards the blade track (dashed line). ( b – e , g – j ) Maximum projection images from confocal z-stacks showing colocalization of marked Gli1 astrocytes (red, b, g ) and S100β or GFAP (green, c , h ). Tissues counterstained with DAPI (blue, d , i) . Merged image shown in ( e , j ). ( k ) Low power micrograph of tomato (red) and BrdU (green) labeled cells at the lesion site showing marked astrocytes at the lesion site proliferating. ( l – o ) Maximum projection images from a confocal stack showing colocalization of tomato (red, l) and BrdU (green, m). Tissues counterstained with DAPI (blue, n). Merged image shown in ( o ). ( p ) Single cell analysis of the proportion of GFAP and Gli1 cells that are colabeled ( n = 1,527 and n = 990 cells, respectively, from 3 animals). Error bars represent mean ± SEM. ( q ) Stereological estimates of cell body volume of marked cells in contralateral and ipsilateral hemispheres. Data points represent individual cells, bars represent mean ± SEM. Statistical signficance was assessed by unpaired Student’s t-test ( p < 0.0001, n = 131 and n = 108 cells, respectively from 4 animals). Scale bars, 250 µm ( a , f , k ), 25 µm ( b – e , g – j , l – o ).

Article Snippet: The following antibodies were used: rabbit anti-S100β (1:40k, DAKO), rabbit anti-βgal (1:40k, MP Bio), rabbit anti-GFAP (1:20k, DAKO), rabbit anti-RFP (1:5k, MBL), rat anti-CD45 (1:4k, BDBiosciences), mouse anti-NeuN (1:5k, Millipore), and sheep anti-BrdU (1:2k, Maine Biotechnology Services).

Techniques: Fluorescence, Immunohistochemistry, Labeling, Single-cell Analysis