Journal: Cell reports

Article Title: Inappropriate Intrusion of an Axonal Mitochondrial Anchor into Dendrites Causes Neurodegeneration

doi: 10.1016/j.celrep.2019.09.012

Figure Lengend Snippet: (A–F) Immunohistochemistry technique for capturing SNPH intrusion in PC dendrites. Shown is SNPH (green), Syt2 (red), and Calbindin (blue) labeling in 3.5-month-old WT (A, C, and E) and Shi (B, D, and F) mice. Scale bar, 10 μm. (G and H) High magnification of the maximum intensity projection image from the z stack through dendritic regions of WT (G) and Shi (H). (I and J) Orthogonal (slice) view of SNPH punctum (indicated by arrows in panels G and H) in the dendritic region from WT (I) and Shi (J) in x-z and y-z orientations, respectively. (K) Quantification of percentage area occupied by SNPH within the dendritic volume from 3 mice of each group. Data are shown as mean ± SEM. *p < 0.05. (L–N) Capturing SNPH intrusion by pre-tagging dendritic mitochondria in vivo using viral transduction. (L) Technique to selectively transduce PCs with AAV-Mito-mCherry. (M) Demonstration of successful pre-tagging of mitochondria in dendrites of a single PC by Calbindin staining. Scale bar, 10 μm. (N) Demonstration of how pre-tagged dendritic mitochondria in PCs are used to capture SNPH intrusion by 3D rotation. (O and P) Single dendritic tree in WT (O) or Shi (P) pre-tagged with Mito-mCherry (red) and SNPH intrusion (green) captured by co-rotation with Mito-mCherry. Merged images show the fraction of dendritic mitochondria anchored by intruded SNPH (yellow). (Q and R) Percentage of PCs with SNPH intrusions (Q) and percentage of SNPH bound to mitochondria per dendritic tree (R) from WT (n = 73) and Shi (n = 106) PCs. Data are shown as mean ± SEM. ***p < 0.005.

Article Snippet: Polyclonal Calbindin , Boster , M03047–2.

Techniques: Immunohistochemistry, Labeling, In Vivo, Transduction, Staining

Journal: Cell reports

Article Title: Inappropriate Intrusion of an Axonal Mitochondrial Anchor into Dendrites Causes Neurodegeneration

doi: 10.1016/j.celrep.2019.09.012

Figure Lengend Snippet: (A and B) Representative images of lentivirally transduced GFP-SNPH (1–469) (A) and GFP-SNPH (B) in PCs of SNPH-KO mice injected with saline (no harmaline, vehicle only). (C-H) Effect of harmaline on GFP-SNPH (1–469)-transduced (C) and GFP-SNPH-transduced (F) PC dendrites. Degenerating dendrites in GFP-SNPH-transduced PCs can be seen in (F). Also shown is Calbindin labeling of GFP SNPH (1–469) (D) and GFP-SNPH (G) from (C) and (F). Merged images of GFP SNPH (1–469) and GFP-SNPH with Calbindin are shown in (E) and (H), respectively. (I–K) Representative image of a harmaline-induced degenerating PC (white arrow in I) transduced with GFP-SNPH. Calbindin staining from the same section is shown in (J), whereas a merged image is shown in (K). Scale bars, 20 μm. (L) Quantification of dendritic shrinkage in GFP-SNPH (1–469)- and GFP-SNPH-transduced PCs in the absence (n = 3 mice, vehicle only) or presence of harmaline (n = 5 mice). Data are shown as mean ± SEM. ***p < 0.001.

Article Snippet: Polyclonal Calbindin , Boster , M03047–2.

Techniques: Injection, Saline, Labeling, Transduction, Staining

Journal: Cell reports

Article Title: Inappropriate Intrusion of an Axonal Mitochondrial Anchor into Dendrites Causes Neurodegeneration

doi: 10.1016/j.celrep.2019.09.012

Figure Lengend Snippet:

Article Snippet: Polyclonal Calbindin , Boster , M03047–2.

Techniques: Virus, Plasmid Preparation, Recombinant, Software, Imaging

Journal: The Journal of Neuroscience

Article Title: Sex-Dependent Regulation of Aromatase-Mediated Synaptic Plasticity in the Basolateral Amygdala

doi: 10.1523/JNEUROSCI.1532-16.2016

Figure Lengend Snippet: AROM expression in major subregions of rodent amygdala. A, Coronal section through anterior amygdala of an young adult mouse, immunostained for AROM using rabbit polyclonal antibodies (Yague et al., 2006). Substantial AROM expression is detectable in the MeA and CeA, in the BL and La nucleus of the BLA, and in the adjacent piriform cortex (Pir). Virtually, no AROM immunoreactivity is seen in the BM of BLA. Low levels of expression were found in the cortical amygdala (CoA). Scale bar, 250 μm. B, Western blots showing AROM protein expression in amygdala subregions CeA, BLA, and MeA of juvenile male and female rats using monoclonal antibodies against AROM (Acris). Tissue from hippocampus (Hip), cerebellum (Cer), and somatosensory cortex (Cor) was blotted for comparison. The strongest signal is found in the MeA. AROM expression levels in the CeA and BLA are comparable to levels in the hippocampus, neocortex, and cerebellum (note: a representative band for the female CeA was inserted from a different gel). C, Quantitative analysis of Western blot data, comparing AROM expression in BLA of age-matched (P20–P24) juvenile male and female rats. No difference between the sexes was evident (rel. expression of AROM: 0.46 ± 0.06 in females; 0.48 ± 0.09 in males; p = 0.88; n = 8 of each sex).

Article Snippet: Rabbit polyclonal anti-calbindin D28K antibodies (Calb; 1:2000; Merck Millipore, Billerica, MA, USA, Cat# AB1778, RRID:AB_2068336) were applied for characterization of the cultures.

Techniques: Expressing, Western Blot, Bioprocessing, Comparison

Journal: The Journal of Neuroscience

Article Title: Clustered Fine Compartmentalization of the Mouse Embryonic Cerebellar Cortex and Its Rearrangement into the Postnatal Striped Configuration

doi: 10.1523/JNEUROSCI.1710-12.2012

Figure Lengend Snippet: Antibody specification

Article Snippet: , Calbindin-D28k , Synthetic peptide derived from amino acids 185-199 of human Calbindin-D-28K , AnaSpec, rabbit polyclonal, Cat. # 53283, Lot # GL141 , 1:4000.

Techniques: Purification, Derivative Assay, Recombinant, Plasmid Preparation

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Early construction of the thalamocortical axon pathway requires c-Jun N-terminal kinase signaling within the ventral forebrain.

doi: 10.1002/dvdy.416

Figure Lengend Snippet: FIGURE 1 Subsets of axon tracts are missing or misrouted in the cTKO brain. A-F, L1 labeling in coronal (A-D) and axial (E-F) sections of E17.5 brains. The internal capsule (arrow) is present in control (A,E) but not cTKO (B,F) brains. An ectopic “U-shaped” bundle projects into the hypothalamus of cTKO brains (open arrowheads, B). The corpus callosum (arrows) and anterior commissure (closed arrowheads) both cross the midline in control (C,E) and cTKO (D,F) brains. G,H, Immunohistochemistry for calbindin in E15.5 brain sections labels a population of axons in the cortical wall of control brains (open arrowheads, G), which are missing from cTKO cortices (asterisks, H). I-R, E15.5 cortices labeled with L1 and Tag1. L1- and Tag1-positive axons are present in control (I-L) and cTKO (N-Q) cortices. L1-positive axons are reduced in cTKO cortices (M); however, Tag1-positive axons are similar between genotypes (R). A population of L1-positive/ Tag1-negative axons is present in control brains (open arrowheads, J-L), but missing in cTKO brains (asterisks, O-Q). AC, anterior commissure; Ctx, cortex; CC, corpus callosum; IC, internal capsule, Thalamus; Tel, telencephalon; Di, diencephalon. Scale bars: A-B, 500 μm; C-F, 300 μm; G,H, 100 μm; I,N, 200 μm; J-L,O-Q, 100 μm

Article Snippet: Primary antibodies: Host Antigen Concentration Company Product number Rabbit Calbindin 1:2000 Swant CB38 Rabbit Darpp-32 1:1000 Synaptic Systems 382 002 Chicken GFP 1:1500 Abcam ab13970 Goat Islet1 1:500 R&D Systems AF1837 Rabbit L1 1:250 Aviva Systems Biology ARP63103 Goat NetrinG1 1:250 R&D Systems AF1166 Rabbit Nkx2.1 1:500 Santa Cruz sc-13 040 Rabbit p-JNK 1:1000 Promega V7931 Mouse Tag1 1:12.5 DSHB AB_2315433 Chicken TH 1:1000 Aves Labs TYH Secondary antibodies: Antibody Concentration Company Product number Donkey anti- Chicken 488 1:4000 Jackson Immuno Research 703-545-155 Goat anti- Chicken 488 1:4000 Invitrogen A11039 Donkey anti- Goat 546 1:2000 Invitrogen A11056 Goat anti- Chicken 546 1:2000 Invitrogen A11040 Goat anti- Mouse 546 1:2000 Invitrogen A11003 Goat anti- Rabbit 546 1:2000 Invitrogen A11010 Donkey anti- Chicken 647 1:2000 Jackson Immuno Research 703-605-155 Donkey anti- Rabbit 647 1:2000 Jackson Immuno Research 711-605-152 Goat anti- Rabbit 647 1:2000 Invitrogen A21244

Techniques: Labeling, Control, Immunohistochemistry