rabbit anti trip8b antibody  (Vector Laboratories)

Journal: PLoS ONE

Article Title: Regulation of Axonal HCN1 Trafficking in Perforant Path Involves Expression of Specific TRIP8b Isoforms

doi: 10.1371/journal.pone.0032181

Figure Lengend Snippet: a–c) Representative low power images of coronal sections of adult wildtype mouse brain show the characteristic distribution of TRIP8b (a) and HCN1 (b) immunoreactivity in hippocampus, with HCN1 and TRIP8b staining most intense in stratum lacunosum-moleculare (SLM) of CA1, and low expression in stratum pyramidale (SP), stratum oriens (SO) and stratum radiatum (SR). In contrast, much lower levels of HCN1 and TRIP8b are expressed in hippocampal dentate gyrus (DG). d–f) Interestingly, in adult TRIP8b −/− mice the immunoreactivity of HCN1 is increased in the MML of DG (arrows in e), where the granule cells are innervated by axons from medial EC via the perforant path. No changes in HCN1 expression were observed in mossy fibres (empty circles), whereas there is significant reduction of HCN1 staining in the SLM of CA1 (asterisks). g–i) Adult TRIP8b[1b/2] −/− mice expressing TRIP8b isoforms 1a and 1a-4 show similar HCN1 staining patterns to wildtype mice in all hippocampal subregions. Scale bar: 200 µm. j–l) Higher magnification views of HCN1 immunoreactivity in TRIP8b −/− DG clearly show the increased HCN1 staining in MML (k) as compared with wildtype (j) and TRIP8b[1b/2] −/− mice (l). Scale bar: 50 µm. m–n) HCN1 immunofluorescence was measured as a function of distance across the superior blade of the dentate gyrus as indicated in the diagram (m), and demonstrates higher HCN1 expression in MML in TRIP8b −/− (red circles) as compared to wildtype (black circles) and TRIP8b[1b/2] −/− (blue circles) mice (n). OML - outer molecular layer, MML - middle molecular layer, IML - inner molecular layer, GCL - granule cell layer, Hil - hilus. Asterisks in (n) denote statistical significance (*p<0.05, **p<0.01, ***p<0.001).

Article Snippet: For light microscopy (TRIP8b expression in rat), sections were incubated with rabbit anti-TRIP8b antibody (1∶10,000 ) overnight, labeled with biotin-conjugated goat anti-rabbit IgG secondary antibody and then subjected to biotin-avidin-peroxidase-complex solution (ABC-Kit, Vector Laboratories).

Techniques: Staining, Expressing, Immunofluorescence

Journal: PLoS ONE

Article Title: Regulation of Axonal HCN1 Trafficking in Perforant Path Involves Expression of Specific TRIP8b Isoforms

doi: 10.1371/journal.pone.0032181

Figure Lengend Snippet: a) Light micrographs of silver-intensified immunogold-stained hippocampal slices show the presence of a thin band of HCN1 immunoreactivity in MML in the TRIP8b −/− (arrows), but not in the wildtype mice. Note also the lack of HCN1 enrichment in stratum lacunosum-moleculare (SLM, asterisks) of CA1 in the TRIP8b −/− -section, as described by Lewis et al. (see also ). b, c) Electron micrographs of serial sections showing immunopositive axonal boutons in a TRIP8b −/− mouse, making both perforated (arrow) and nonperforated synapses with dendritic spines in MML. Abbreviations: at, axon terminal; sp, spine. d) Percentage of axonal boutons, immunopositive for HCN1, making axospinous synapses in MML in wildtype (black) and TRIP8b −/− mice (white). e) Average number of particles per synaptic bouton in wildtype (black) and TRIP8b −/− mice (white). Data were obtained from two mice of each genotype, and based on analyses of 25 synaptic boutons from each mouse (100 axonal boutons total) from the MML. Serial ultrathin sections were obtained from slices similar to those shown in (a).

Article Snippet: For light microscopy (TRIP8b expression in rat), sections were incubated with rabbit anti-TRIP8b antibody (1∶10,000 ) overnight, labeled with biotin-conjugated goat anti-rabbit IgG secondary antibody and then subjected to biotin-avidin-peroxidase-complex solution (ABC-Kit, Vector Laboratories).

Techniques: Staining

Journal: PLoS ONE

Article Title: Regulation of Axonal HCN1 Trafficking in Perforant Path Involves Expression of Specific TRIP8b Isoforms

doi: 10.1371/journal.pone.0032181

Figure Lengend Snippet: a) Horizontal section showing hippocampus, entorhinal and perirhinal cortex in an adult rat, immunostained (IHC) for TRIP8b. Note: TRIP8b immunoreactivity is strong in areas which also show substantial HCN1 expression : the distal dendritic fields of CA1 and subiculum (Sub) and the outer layers (I–III) of medial entorhinal cortex (medEC, arrows). TRIP8b expression is further visible in deep layer (V) of both the medial and the lateral EC, containing pyramidal cells. In the perirhinal cortex (peCo), this staining is replaced by TRIP8b immunoreactivity in the outermost layers (asterisks), most likely reflecting dendritic positioning of the TRIP8b in the pyramidal cells. b) Higher magnification view of medial EC from a): TRIP8b immunoreactivity is limited to layers I–III and V, but diffuse immunostaining does not permit identification of the TRIP8b-expressing neurons. c) Non-radioactive in situ hybridization (ISH) revealed expression of TRIP8b mRNA specifically in layers II and V, indicating that the stellate cells of layer II and the pyramidal cells of layer V are the major sources of TRIP8b immunoreactivity in medial EC. d–f) Co-labeling of TRIP8b mRNA (d) and reelin protein (e) further showed that many of the TRIP8b mRNA-positive cells in EC layer II co-express reelin (f), identifying them as perforant path projection neurons . Scale bar: 500 µm (a), 200 µm (b, c), 80 µm (d–f). g) Upper panel: Western Blots illustrating expression of TRIP8b and HCN1 in EC tissue of immature (P10) and adult (>P60) rats. As shown previously , , probing with a pan-TRIP8b antibody revealed two bands, representing predominantly the TRIP8b(1a) and (1a-4) isoforms (∼65 and ∼70 kDa, respectively), which together constitute >80% of cortical TRIP8b. Lower panel: Native TRIP8b in EC (left) run as a control against TRIP8b(1a) (right) and TRIP8b(1a-4) (middle) harvested from HEK293 cells that were single-transfected with those isoforms. h) Quantitative analysis of HCN1 and TRIP8b isoform expression levels in immature vs. adult EC. Data are presented as “% of immature” expression and have been normalized to actin. Expression of both the TRIP8b(1a) (245±41%) and the (1a-4) isoform (200±30%) increased significantly with age (p<0.01 for both), whereas no significant change was detected for HCN1 (140±17%, p>0.05).

Article Snippet: For light microscopy (TRIP8b expression in rat), sections were incubated with rabbit anti-TRIP8b antibody (1∶10,000 ) overnight, labeled with biotin-conjugated goat anti-rabbit IgG secondary antibody and then subjected to biotin-avidin-peroxidase-complex solution (ABC-Kit, Vector Laboratories).

Techniques: Expressing, Staining, Immunostaining, In Situ Hybridization, Labeling, Western Blot, Transfection

Journal: PLoS ONE

Article Title: Regulation of Axonal HCN1 Trafficking in Perforant Path Involves Expression of Specific TRIP8b Isoforms

doi: 10.1371/journal.pone.0032181

Figure Lengend Snippet: Confocal images showing representative neurons in “immature cultures” (P0+4 days in vitro ) single-transfected with either HCN1-EGFP (a–c), TRIP8b(1a-4) (d-f) or TRIP8b(1a) (g–i). For the identification of axons, the microtubule-associated protein Tau-1 was co-labeled (b, e, h). Note: Single-transfection resulted in a relatively homogeneous distribution of the overexpressed proteins within neuronal compartments, including the axon (arrows). Scale bar: 20 µm.

Article Snippet: For light microscopy (TRIP8b expression in rat), sections were incubated with rabbit anti-TRIP8b antibody (1∶10,000 ) overnight, labeled with biotin-conjugated goat anti-rabbit IgG secondary antibody and then subjected to biotin-avidin-peroxidase-complex solution (ABC-Kit, Vector Laboratories).

Techniques: In Vitro, Transfection, Labeling

Journal: PLoS ONE

Article Title: Regulation of Axonal HCN1 Trafficking in Perforant Path Involves Expression of Specific TRIP8b Isoforms

doi: 10.1371/journal.pone.0032181

Figure Lengend Snippet: a–h) Confocal images showing representative neurons in “immature cultures” that co-express HCN1-EGFP (a, e) and TRIP8b(1a-4) (b, f). Note: Distribution of both proteins is relatively uniform within the neurons and their expression can be detected over long range within axons (arrows) labeled with Tau-1 (c, d, g, h). i–p) Confocal images showing representative neurons in “immature cultures” that co-express HCN1-EGFP (i, m) and TRIP8b(1a) (j, n). Both proteins are not uniformly distributed in these neurons, but appear enriched in soma and proximal dendrites (arrowheads in i, j, m, n), while expression in axons (k, l, o, p; arrows) is low (m–p) or absent (i–l). Scale bar: 20 µm. r–t) Quantitative analysis of the relative subcellular distribution of HCN1-EGFP- and TRIP8b-signal in neurons co-transfected with either TRIP8b(1a) or TRIP8b(1a-4). Neuronal area was divided into four subdivisions (each 100 µm 2 ) with subdivision 1 containing the axon hillock (r, arrows). Signal intensity was determined in each subdivision and relative signal intensity was calculated by dividing each subdivisional value by the mean of all four values (“% of mean”). Values from 1a-4- and 1a-transfected neurons (n = 25 each) were then compared. Note: While HCN1-EGFP and TRIP8b distribution showed a preference towards the somatodendritic subdivision 3 in both the TRIP8b(1a) and the TRIP8b(1a-4)-co-transfected neurons, enrichment in this subdivision was significantly more pronounced in neurons co-transfected with TRIP8b(1a) (HCN1: 193±14 vs. 150±12%; TRIP8b: 178±15 vs. 136±15%; p = 0.02 and 0.04, respectively). In contrast, relative signal intensities for HCN1-EGFP and TRIP8b in subdivision 1, containing the axon hillock, were significantly lower in TRIP8b(1a)- compared to TRIP8b(1a-4) co-transfected neurons (HCN1: 29±4 vs. 51±5%; TRIP8b: 36±5 vs. 70±8%; p = 0.002 and p = 0.0009, respectively).

Article Snippet: For light microscopy (TRIP8b expression in rat), sections were incubated with rabbit anti-TRIP8b antibody (1∶10,000 ) overnight, labeled with biotin-conjugated goat anti-rabbit IgG secondary antibody and then subjected to biotin-avidin-peroxidase-complex solution (ABC-Kit, Vector Laboratories).

Techniques: Expressing, Labeling, Transfection

Journal: PLoS ONE

Article Title: Regulation of Axonal HCN1 Trafficking in Perforant Path Involves Expression of Specific TRIP8b Isoforms

doi: 10.1371/journal.pone.0032181

Figure Lengend Snippet: a–d) Confocal images showing a representative neuron in “differentiated cultures” (P5+10 days in vitro ) that co-expresses endogenous HCN1 (a) and TRIP8b(1a-4) (b). Expression of both proteins is relatively homogenous within the neuron, involving soma, dendrites and the axon (labeled with Tau, arrows; c, d). e–h) Confocal images showing a representative neuron in “differentiated cultures” that co-expresses emdogenous HCN1 (e) and TRIP8b(1a) (f). While expression of both proteins can be observed in all compartments, including the axon (g, h), expression seems to be most intense in the somatodendritic compartment (arrowheads). Scale bar: 20 µm. i–k) Quantitative analyses of the relative subcellular distribution of endogenous HCN1- and TRIP8b-signal in differentiated entorhinal neurons transfected with either TRIP8b(1a) or TRIP8b(1a-4) (n = 25 each). Data from TRIP8b(1a-4)–overexpressing neurons confirm a relatively homogenous distribution of both the HCN1 and TRIP8b throughout the neurons (j, k). In contrast, in neurons transfected with TRIP8b(1a), TRIP8b was preferentially expressed in somatodendritic subdivision 3 (153±12 in 1a- vs. 119±10% in 1a-4-expressing neurons; p = 0.04; k). HCN1 expression in these neurons showed a trend towards enrichment in the somatodendritic compartment, which was, however, not significant (subdivision 1: 63±12 in 1a- vs. 88±12% in 1a-4-expressing neurons; p = 0.14; subdivision 3: 150±14 in 1a- vs. 129±13% in 1a-4-expressing neurons; p = 0.24; j).

Article Snippet: For light microscopy (TRIP8b expression in rat), sections were incubated with rabbit anti-TRIP8b antibody (1∶10,000 ) overnight, labeled with biotin-conjugated goat anti-rabbit IgG secondary antibody and then subjected to biotin-avidin-peroxidase-complex solution (ABC-Kit, Vector Laboratories).

Techniques: In Vitro, Expressing, Labeling, Transfection

Journal: Molecular Psychiatry

Article Title: Glucocorticoid-glucocorticoid receptor-HCN1 channels reduce neuronal excitability in dorsal hippocampal CA1 neurons

doi: 10.1038/s41380-022-01682-9

Figure Lengend Snippet: A Illustration depicting the acute corticosterone treatment in the dorsal hippocampus. B Representative dorsal hippocampal slices immunolabeled with antibody against TRIP8b. Rectangle boxes depict the region of the slice used for quantification of the optical density. The arrows indicate increased perisomatic TRIP8b protein expression. C Quantification of TRIP8b protein expression from the perisomatic region to the distal dendritic region of CA1 from the dorsal hippocampus. D Representative dorsal hippocampal slices immunolabeled with antibody against HCN1. Rectangle boxes depict the region of the slice used for quantification of the optical density. The arrows indicate increased perisomatic HCN1 protein expression. E Quantification of HCN1 protein expression from the perisomatic region to the distal dendritic region of CA1 from the dorsal hippocampus. F , G We performed cell-attached voltage-clamp recordings. F Representative maximal h current traces in response to a 500-ms hyperpolarizing voltage step (−140 mV). G I h was significantly elevated in the dorsal CA1 neurons from corticosterone treatment compared with those from the vehicle-treated group. Dexamethasone reduced R in at RMP ( H ) and at −65 mV ( J ) in dorsal CA1 neurons. Corticosterone-induced decrease in R in at RMP ( H ) and at −65 mV ( J ) was blocked by RU 486, KT5720, and ZD7288. Changes in R in at RMP ( I ) and at −65 mV ( K ) were blocked by RU 486, KT5720, and ZD7288. Data are expressed as mean ± SEM.

Article Snippet: Primary antibody in this study was used as follows; rabbit-anti-HCN1 (1:500, Invitrogen, Cat # PA5-78675), rabbit anti-HCN2 (1:500, Invitrogen, Cat # PA1-918), rabbit anti-HCN3 (1:500, Invitrogen, Cat # PA5-104434), rabbit anti-HCN4 (1:500, Invitrogen, Cat # PA5-111793), rabbit C-terminal TRIP8b (1:500, Proteintech, Cat #13084-1-AP), and rabbit anti-GR (1:500, Invitrogen, Cat # PA1-511A).

Techniques: Immunolabeling, Expressing

Journal: Molecular Psychiatry

Article Title: Glucocorticoid-glucocorticoid receptor-HCN1 channels reduce neuronal excitability in dorsal hippocampal CA1 neurons

doi: 10.1038/s41380-022-01682-9

Figure Lengend Snippet: A Timeline of CSDS, behavioral tests, electrophysiology, and biochemical assay. B CSDS produced the susceptible and resilient phenotype during the social interaction test. After 1 month ( C ) or 3 months ( D ) of no CSDS, susceptible mice showed persistent social avoidance during the social interaction test. E , F We performed whole-cell current-clamp recordings. E Representative voltage responses with depolarizing current step (210 pA; 750 ms) at RMP in dorsal CA1 neurons. F Dorsal CA1 neurons of susceptible group had lower action potential firing than control group, whereas the resilient group had higher action potential firing. G , H We performed cell-attached voltage-clamp recordings. G Representative maximal h current traces in response to a 500-ms hyperpolarizing voltage step (−140 mV). H I h was significantly elevated in the dorsal CA1 neurons from susceptible group compared with those from the control and resilient mice. Representative dorsal hippocampal slices immunolabeled with antibody against HCN1 ( I ) and TRIP8b ( K ). Rectangle boxes depict the region of the slice used for quantification of the optical density. The arrows indicate increased perisomatic HCN1 and TRIP8b protein expression. Quantification of HCN1 ( J ) and TRIP8b ( L ) protein expression from the perisomatic region to the distal dendritic region of CA1 from the dorsal hippocampus. M , N We performed whole-cell current-clamp recordings. M Corticosterone reduced R in at RMP of the dorsal CA1 neurons in the control and resilient groups, but had no effect on R in in susceptible group. N Changes in R in at RMP were much lower in susceptible group compared to the control and resilient groups. Data are expressed as mean ± SEM.

Article Snippet: Primary antibody in this study was used as follows; rabbit-anti-HCN1 (1:500, Invitrogen, Cat # PA5-78675), rabbit anti-HCN2 (1:500, Invitrogen, Cat # PA1-918), rabbit anti-HCN3 (1:500, Invitrogen, Cat # PA5-104434), rabbit anti-HCN4 (1:500, Invitrogen, Cat # PA5-111793), rabbit C-terminal TRIP8b (1:500, Proteintech, Cat #13084-1-AP), and rabbit anti-GR (1:500, Invitrogen, Cat # PA1-511A).

Techniques: Produced, Control, Immunolabeling, Expressing