Journal: Antioxidants

Article Title: Nrf2 Activation Does Not Protect from Aldosterone-Induced Kidney Damage in Mice

doi: 10.3390/antiox12030777

Figure Lengend Snippet: Localization of the activated transcription factor Nrf2. Representative pictures of the Western blots of the expression of total Nrf2 phosphorylated at Ser40 (pNrf2, 110 kDa) in ( a ) nuclear and ( b ) cytosolic extracts of kidneys of mice as well as the quantification of band densities of the above-mentioned protein measured via ImageJ and related to the housekeepers lamin B2 (68 kDa) and GAPDH (37 kDa). Percentage of pNrf2-positive stained nuclei in cortex ( c ) and medulla ( d ). For the quantification of positive Nrf2 nuclei, 10 visual fields of cortical and 3–5 visual fields of medullary kidney sections were analyzed per animal via ImageJ. ( e ) Representative images of double stained kidney sections used for the localization of pNrf2 in kidney cells. Double staining on paraffin-embedded kidney sections was carried out using antibodies against pNrf2 (brown staining) and against calbindin (purple staining), a marker for distal tubule and early collecting duct cells. Examples of pNrf2-positive stained nuclei are indicated by black arrows; white arrows indicate the corresponding section of the tubulus system. Proximal tubules were identified by the presence of the brush border, whereas glomeruli were identified by their capillary tuft (blue circles). Distal tubules and the early collecting duct were visualized by positive calbindin staining. The late collecting duct was identified by the absence of positive calbindin staining and brush border. ( f – i ) Quantification of pNrf2-positive nuclei in the four kidney structures related to the number of nuclei in regions positive for the specific kidney cell identifiers in 10 visual fields. For the quantification in the glomerulus, 50 glomeruli were evaluated. Aldo: aldosterone, C: control, Nrf2: nuclear factor erythroid 2-related factor 2, Sulf: sulforaphane, WT: wild type. Data are shown as mean + SEM, n = 7–8. * p ≤ 0.05 vs. WT-C, # p ≤ 0.05 vs. WT-Sulf, ° p ≤ 0.05 vs. WT-Aldo, ^ p ≤ 0.05 vs. Nrf2ꜛ-C.

Article Snippet: After visualization of antibody binding, the protocol was repeated with an antibody against calbindin (1:200, #2173, Cell Signaling, Herts, UK) to identify distal tubular cells and cells of the early collecting duct.

Techniques: Western Blot, Expressing, Staining, Double Staining, Marker, Control

Journal: Neurobiology of disease

Article Title: Human IPSC 3D brain model as a tool to study chemical-induced dopaminergic neuronal toxicity.

doi: 10.1016/j.nbd.2022.105719

Figure Lengend Snippet: Fig. 1. Experimental design and BrainSpheres characterization. A) shows diagram of experimental procedure described in material and methods. B) shows confocal images of neuronal marker BTUBIII, dopaminergic neurons markers (KCNJ6, CALB1), astrocytes marker (GFAP) and oligodendrocyte marker (O4). Bars represent 50 μm.

Article Snippet: Subsequently, the BrainSpheres were washed twice with PBS (1×) and incubated with blocking solution (10% goat serum, 1% BSA, 0.15% saponin in PBS) at 4 ◦C for 1 h. The BrainSpheres were washed with washing solution (1% BSA, 0.15% saponin in PBS), and incubated overnight at 4 ◦C with primary antibodies: anti- Calbindin 1 (CALB1) (1:200, for dopaminergic neurons) [ABclonal], anti-Potassium Inwardly Rectifying Channel Subfamily J Member 6 (KCNJ6) (1:200, for dopaminergic neurons)[ABclonal], anti-tyrosine hydroxylase (TH) (1:100, for dopaminergic neurons)[Millipore], anti-NF200 (1:200, neurofilament for all neurons) [Sigma] and anti-ZO-1 (1:200, endothelial tight junctions) [Life Technologies] diluted in blocking solution.

Techniques: Marker

Journal: Neurobiology of disease

Article Title: Human IPSC 3D brain model as a tool to study chemical-induced dopaminergic neuronal toxicity.

doi: 10.1016/j.nbd.2022.105719

Figure Lengend Snippet: Fig. 4. Alteration in expression of dopaminergic neurons- and oxidative stress-related genes and extracellular levels of metabolites after 24 h treatment of Brain Spheres with 6-OHDA (500 μM), MPTP (5000 μM) and MPP+ (1000 μM). Gene expression of markers for dopaminergic neurons (TH, SLC6A3, CALB1, KCNJ6, TBR1), mitochondrial metabolism (ATP5O, NDUFB1, NDUFA1) and oxidative stress (GSTO1, KEAP1, SOD2) was analyzed by real-time qPCR. B) Extracellular metabolites measured by mass spectrometry (LC-MS/MS). C) Gene expression after compound washout and 5 days recovery period.In B and C bars represent mean ± SEM of 3–4 independent experiments. One-Way ANOVA followed by Kruskal–Wallis multiple comparison H tests was performed to evaluate statistical significance (* p < 0.05, ** p < 0.005, *** p < 0.0005, **** P < 0.0001).

Article Snippet: Subsequently, the BrainSpheres were washed twice with PBS (1×) and incubated with blocking solution (10% goat serum, 1% BSA, 0.15% saponin in PBS) at 4 ◦C for 1 h. The BrainSpheres were washed with washing solution (1% BSA, 0.15% saponin in PBS), and incubated overnight at 4 ◦C with primary antibodies: anti- Calbindin 1 (CALB1) (1:200, for dopaminergic neurons) [ABclonal], anti-Potassium Inwardly Rectifying Channel Subfamily J Member 6 (KCNJ6) (1:200, for dopaminergic neurons)[ABclonal], anti-tyrosine hydroxylase (TH) (1:100, for dopaminergic neurons)[Millipore], anti-NF200 (1:200, neurofilament for all neurons) [Sigma] and anti-ZO-1 (1:200, endothelial tight junctions) [Life Technologies] diluted in blocking solution.

Techniques: Expressing, Gene Expression, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Comparison

Journal: International Journal of Molecular Sciences

Article Title: RNA-Seq Analysis Reveals an Essential Role of the Tyrosine Metabolic Pathway and Inflammation in Myopia-Induced Retinal Degeneration in Guinea Pigs

doi: 10.3390/ijms222212598

Figure Lengend Snippet: Horizontal cells and amacrine cells in different groups of retinas. ( A ) Representative confocal images of GABA labelled retinas from PP of naïve control, self-control and FDM eyes. Nuclei were stained with DAPI (blue). ( B ) Quantitative data showing the number of GABAergic amacrine cells (INL) in different retinal locations of the three groups guinea pigs. ( C ) Representative confocal images of CHAT (red) and Calbindin (green) labelled retinas from PP of naïve control, self-control and FDM eyes. Nuclei were stained with DAPI (blue). ( D , E ) Quantitative data showing the number of CHATergic amacrine cells ( D ) and calbindin + horizontal cells in different retinal locations of the three groups of guinea pigs. INL, inner nuclear layer; ONL, outer nuclear layer. Scale Bar: 50 µm. Mean ± SD, n = 3~9/group. * p < 0.05. One-way ANOVA followed by Fisher’s LSD test.

Article Snippet: Calbindin , Rabbit , TA342845 , 1:50 , Origene , Horizontal cells.

Techniques: Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: RNA-Seq Analysis Reveals an Essential Role of the Tyrosine Metabolic Pathway and Inflammation in Myopia-Induced Retinal Degeneration in Guinea Pigs

doi: 10.3390/ijms222212598

Figure Lengend Snippet: Primary and secondary antibodies used in immunofluorescence study.

Article Snippet: Calbindin , Rabbit , TA342845 , 1:50 , Origene , Horizontal cells.

Techniques: Immunofluorescence, Plasmid Preparation

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