primary antibody concentration source  (Bio-Rad)

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: NECAB1 and NECAB2 are Prevalent Calcium-Binding Proteins of CB1/CCK-Positive GABAergic Interneurons.

doi: 10.1093/cercor/bhaa326

Figure Lengend Snippet: Figure 1. Single-cell RNA-Seq profile of representative calcium-binding proteins reveals high Necab1 and Necab2 expression in CB1-receptor-expressing GABAergic interneurons. (a, b) In silico analysis of selected calcium-binding protein encoding mRNA expression profiles was performed on data derived from a publicly available single-cell RNAseq database (Zeisel et al. 2015). The samples were obtained from the mouse hippocampus (a) and the somatosensory cortex (b). Cells in the database were selected for their GABAergic phenotype and high Cnr1 expression to focus the analysis on CB1/CCK-positive interneurons. The mRNAs of characteristic EF-hand calcium-binding proteins (calbindin—Calb1, calretinin—Calb2, parvalbumin—Pvalb and secretagogin—Scgn) are present in low levels in most of these interneurons, and only a few of them exhibit elevated Calb1 levels. In contrast, the genes encoding the N-terminal EF-hand calcium-binding protein 1 and 2 (Necab1 and Necab2, respectively) are consistently expressed at high levels in all investigated hippocampal and cortical interneurons (n = 61 cells in the hippocampus, n = 84 cells in the somatosensory cortex).

Article Snippet: 31, No. 3 Antibody type Target Species Concentration Source Primary antibodies Anti-CB1 Guinea pig 1:2000 Fukudome et al. (2004) Anti-NECAB1 Rabbit 1:300 Atlas Antibodies, HPA023629 Anti-NECAB2 Rabbit 1:500 Atlas Antibodies, HPA013998 Anti-CCK Mouse 1:3000 CURE, 39161 Anti-GAD67 Mouse 1:2000 Merck, MAB5406 Anti-PV Mouse 1:5000 Swant, 235 Secondary antibodies Anti-guinea pig, Alexa 488-conjugated Donkey 1:400 Jackson, 706-545-148 Anti-rabbit Alexa 594-conjugated Donkey 1:400 Jackson, 711-585-152 Anti-mouse Alexa 488-conjugated Donkey 1:400 Jackson, 715-545-150 Anti-guinea pig Alexa-594-conjugated Donkey 1:400 Jackson, 706-585-148 Anti-rabbit Alexa 647-conjugated Donkey 1:400 Jackson, 711-605-152 Anti-rabbit Alexa 488-conjugated Donkey 1:400 Jackson, 711-545-152 Anti-mouse Alexa 594-conjugated Donkey 1:400 Jackson, 715-585-150 Anti-mouse Alexa 647-conjugated Donkey 1:400 Jackson, 715-605-150 Note: Referred in Materials and Methods/Immunostaining section. sections were covered with 25 μL of freshly prepared Smart Buffer (Abbelight) imaging medium, then sealed with nail polish.

Techniques: RNA Sequencing, Binding Assay, Expressing, In Silico, Derivative Assay

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: NECAB1 and NECAB2 are Prevalent Calcium-Binding Proteins of CB1/CCK-Positive GABAergic Interneurons.

doi: 10.1093/cercor/bhaa326

Figure Lengend Snippet: Figure 3. Prominent Necab1 and Necab2 mRNA expression in high Cnr1-expressing hippocampal, cortical, and amygdalar interneurons. (a–d) Representative confocal microscopy images of triple fluorescent ISH shows the expression patterns of genes encoding the CB1 cannabinoid receptor (Cnr1, yellow), NECAB1 (Necab1, cyan), and NECAB2 (Necab2, green) in the mouse hippocampus (a1–b3), somatosensory cortex (c1–c3), and BLA complex (d1–d3). Cell nuclei are stained with DAPI (white) in all images. (b1–b3) High magnification of a triple-positive hippocampal interneuron located in the CA1 stratum oriens is shown from the boxed region in (a1–a3). Dashed line in (d1–d3) outlines the BLA complex. (e–g) Quantitative analysis of mRNA levels in 1337 individual cells segregates neurons into two major populations throughout the hippocampus (HC), somatosensory cortex (SS), and BLA complex. Cells exhibiting strong (S) Cnr1 mRNA expression are predominantly represent GABAergic interneurons, whereas neurons with weak (W) Cnr1 mRNA levels belong to principal cells. The three columns of data points represent individual cells from n = 3 mice. Note that both Necab1 and Necab2 are consistently highly expressed in strong Cnr1-positive cells in all three regions. Interestingly, weak Cnr1-positive cells that are putative mossy cells in the hilus of the DG also express moderate levels of both Necabs. Specific pyramidal cell populations express Necab1 in the somatosensory cortex and in the BLA complex, whereas Necab2 has high levels in CA2 and CA3a pyramidal neurons.

Article Snippet: 31, No. 3 Antibody type Target Species Concentration Source Primary antibodies Anti-CB1 Guinea pig 1:2000 Fukudome et al. (2004) Anti-NECAB1 Rabbit 1:300 Atlas Antibodies, HPA023629 Anti-NECAB2 Rabbit 1:500 Atlas Antibodies, HPA013998 Anti-CCK Mouse 1:3000 CURE, 39161 Anti-GAD67 Mouse 1:2000 Merck, MAB5406 Anti-PV Mouse 1:5000 Swant, 235 Secondary antibodies Anti-guinea pig, Alexa 488-conjugated Donkey 1:400 Jackson, 706-545-148 Anti-rabbit Alexa 594-conjugated Donkey 1:400 Jackson, 711-585-152 Anti-mouse Alexa 488-conjugated Donkey 1:400 Jackson, 715-545-150 Anti-guinea pig Alexa-594-conjugated Donkey 1:400 Jackson, 706-585-148 Anti-rabbit Alexa 647-conjugated Donkey 1:400 Jackson, 711-605-152 Anti-rabbit Alexa 488-conjugated Donkey 1:400 Jackson, 711-545-152 Anti-mouse Alexa 594-conjugated Donkey 1:400 Jackson, 715-585-150 Anti-mouse Alexa 647-conjugated Donkey 1:400 Jackson, 715-605-150 Note: Referred in Materials and Methods/Immunostaining section. sections were covered with 25 μL of freshly prepared Smart Buffer (Abbelight) imaging medium, then sealed with nail polish.

Techniques: Expressing, Confocal Microscopy, Staining

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: NECAB1 and NECAB2 are Prevalent Calcium-Binding Proteins of CB1/CCK-Positive GABAergic Interneurons.

doi: 10.1093/cercor/bhaa326

Figure Lengend Snippet: Figure 4. NECAB1 and NECAB2 calcium-binding proteins are distributed in CB1-receptor-expressing GABAergic interneurons. (a–l) Representative confocal microscopy images of double immunostaining of CB1 receptors (yellow) and either NECAB1 (cyan) or NECAB2 (green). Cell nuclei are stained with DAPI (white). Interneurons colocalizing CB1/NECAB1 or CB1/NECAB2 stand out already at low magnification in the CA1 subregion of the hippocampus CA1 region (a, c), in the somatosensory cortex (e, g) and in the BLA complex (i, k). (b1–2, d1–2, f1–2, h1–2, j1–2, l1–2) High magnification shows the double-immunostained cell bodies from the respective boxed areas present in a, c, e, g, i, k. Note that both CB1 receptor- and NECAB2-immunostaining visualize a dense meshwork of axons in all three regions, whereas NECAB1 is primarily located in somata and dendrites.

Article Snippet: 31, No. 3 Antibody type Target Species Concentration Source Primary antibodies Anti-CB1 Guinea pig 1:2000 Fukudome et al. (2004) Anti-NECAB1 Rabbit 1:300 Atlas Antibodies, HPA023629 Anti-NECAB2 Rabbit 1:500 Atlas Antibodies, HPA013998 Anti-CCK Mouse 1:3000 CURE, 39161 Anti-GAD67 Mouse 1:2000 Merck, MAB5406 Anti-PV Mouse 1:5000 Swant, 235 Secondary antibodies Anti-guinea pig, Alexa 488-conjugated Donkey 1:400 Jackson, 706-545-148 Anti-rabbit Alexa 594-conjugated Donkey 1:400 Jackson, 711-585-152 Anti-mouse Alexa 488-conjugated Donkey 1:400 Jackson, 715-545-150 Anti-guinea pig Alexa-594-conjugated Donkey 1:400 Jackson, 706-585-148 Anti-rabbit Alexa 647-conjugated Donkey 1:400 Jackson, 711-605-152 Anti-rabbit Alexa 488-conjugated Donkey 1:400 Jackson, 711-545-152 Anti-mouse Alexa 594-conjugated Donkey 1:400 Jackson, 715-585-150 Anti-mouse Alexa 647-conjugated Donkey 1:400 Jackson, 715-605-150 Note: Referred in Materials and Methods/Immunostaining section. sections were covered with 25 μL of freshly prepared Smart Buffer (Abbelight) imaging medium, then sealed with nail polish.

Techniques: Binding Assay, Expressing, Confocal Microscopy, Double Immunostaining, Staining, Immunostaining

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: NECAB1 and NECAB2 are Prevalent Calcium-Binding Proteins of CB1/CCK-Positive GABAergic Interneurons.

doi: 10.1093/cercor/bhaa326

Figure Lengend Snippet: Figure 5. Subcellular distribution of NECAB1 and NECAB2 in identified perisomatically targeting and dendritically targeting CB1-receptor-expressing GABAergic interneurons. (a) MIP of a representative perisomatically targeting multipolar neuron in CA1 stratum radiatum (s.r.). The axonal arbor is restricted to stratum pyramidale (s.p.), whereas the dendrites are extended to stratum oriens (s.o.) and stratum radiatum. Boxed areas depict the different subcellular compartments that are shown at high magnification in b–h with matching colors. The neuron was filled with biocytin during patch-clamp recording. Insets show example voltage traces in response to depolarizing and hyperpolarizing current steps of −200, 0, and +150 pA from resting membrane potential recorded in whole-cell current-clamp configuration (scale: 40 mV, 200 ms). Note the regular-spiking, accommodating firing pattern, a characteristic feature of CB1/CCK-positive GABAergic interneurons. (b1–b2) Indeed, biocytin- filled axon terminals (b1) belonging to the same interneuron carry high density of CB1 receptors (yellow, b2). (c1–d2) The cell body of this interneuron was cut in half during resectioning (c1, d1) that makes the demonstration of the presence of both NECAB1 (cyan) and NECAB2 (green) within the soma possible (c2, d2). (e1–f2) Dendrites of the biocytin-filled cell also contain both NECAB1 (e2) and NECAB2 (f2). (g1–h2) In contrast, confocal imaging reveals intense NECAB2-immunostaining (h2) in axon terminals, whereas NECAB1-immunostaining remained under detection threshold in boutons of the same interneuron (g2). (i–j2) MIP of a confocal z-stack presents a regular-spiking dendritically targeting interneuron with an extensively distributed axonal arbor that contain CB1 receptors (j2). The subcellular distribution of the NECAB calcium-binding proteins within this cell mirrors their localization in the perisomatically targeting interneuron presented in (a). While the cell body and the dendrites contain both NECABs (k1–n2), only NECAB2-immunostaining is observed in the axon terminals (p2).

Article Snippet: 31, No. 3 Antibody type Target Species Concentration Source Primary antibodies Anti-CB1 Guinea pig 1:2000 Fukudome et al. (2004) Anti-NECAB1 Rabbit 1:300 Atlas Antibodies, HPA023629 Anti-NECAB2 Rabbit 1:500 Atlas Antibodies, HPA013998 Anti-CCK Mouse 1:3000 CURE, 39161 Anti-GAD67 Mouse 1:2000 Merck, MAB5406 Anti-PV Mouse 1:5000 Swant, 235 Secondary antibodies Anti-guinea pig, Alexa 488-conjugated Donkey 1:400 Jackson, 706-545-148 Anti-rabbit Alexa 594-conjugated Donkey 1:400 Jackson, 711-585-152 Anti-mouse Alexa 488-conjugated Donkey 1:400 Jackson, 715-545-150 Anti-guinea pig Alexa-594-conjugated Donkey 1:400 Jackson, 706-585-148 Anti-rabbit Alexa 647-conjugated Donkey 1:400 Jackson, 711-605-152 Anti-rabbit Alexa 488-conjugated Donkey 1:400 Jackson, 711-545-152 Anti-mouse Alexa 594-conjugated Donkey 1:400 Jackson, 715-585-150 Anti-mouse Alexa 647-conjugated Donkey 1:400 Jackson, 715-605-150 Note: Referred in Materials and Methods/Immunostaining section. sections were covered with 25 μL of freshly prepared Smart Buffer (Abbelight) imaging medium, then sealed with nail polish.

Techniques: Expressing, Patch Clamp, Membrane, Imaging, Immunostaining, Binding Assay