Journal: Frontiers in Cell and Developmental Biology

Article Title: Maturation of type I and type II rat vestibular hair cells in vivo and in vitro

doi: 10.3389/fcell.2024.1404894

Figure Lengend Snippet: Primary antibodies.

Article Snippet: Calretinin , 1/500 , Guinea pig polyclonal , 214.104, Synaptic Systems, RRID: AB_10635160 , Co-localizes with anti-calretinin antibodies validated in KO mice (CR 7699 from Swant).

Techniques: Western Blot, Control, Expressing, Clinical Proteomics, Membrane, Immunocytochemistry, Knockdown

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: International journal of molecular medicine

Article Title: Aqueous extract of pomegranate seed attenuates glucocorticoid-induced bone loss and hypercalciuria in mice: A comparative study with alendronate.

doi: 10.3892/ijmm.2016.2622

Figure Lengend Snippet: Figure 3. Expression of calcium transport proteins in the kidney. (A and B) The protein levels of calcium transporters [transient receptor potential vanilloid (TRPV)5, TRPV6, calbindin-D9k (CaBP-9k) and plasma membrane Ca2+-ATPase1 (PMCA1)] were measured in the kidney. Values are expressed as the means ± SEM, n=8 in each group. *P<0.05 versus vehicle group; #P<0.05 versus dexamethasone (DXM) group. Vehicle, vehicle group (n=8); DXM group, mice were injected intramuscularly with 5 mg/kg body weight DXM three times/week for 12 weeks (n=8); ALN group, mice rece ived alendronate orally at a dose of 0.1 mg/kg/day in combination with DXM for 12 weeks (n=8); pomegranate seed (PS) group, mice received daily drinks of aqueous extract of pomegranate seed (AE-PS) in combination with DXM for 12 weeks (n=8).

Article Snippet: After saturation with 5% (w/v) non-fat dry milk in Tris-buffered saline (TBS) and 0.1% (w/v) Tween-20 (TBST), the membranes were incubated with the following antibodies: transient receptor potential vanilloid (TRPV)5 (sc-398345; 1:500), TRPV6 (sc-28763; 1:500), osteoprotegerin (OPG; sc-8468; 1:1,000) and receptor activator of nuclear factor-κB ligand (RANKL; sc-7628; 1:2,000) , CaSR (sc-32181; 1:500), calbindin-D9k (CaBP-9k; sc-367381; 1:500), plasma membrane Ca2+-ATPase1 (PMCA1; sc-16488; 1:500) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), overnight at 4 ̊C.

Techniques: Expressing, Clinical Proteomics, Membrane, Injection

Journal: International journal of molecular medicine

Article Title: Aqueous extract of pomegranate seed attenuates glucocorticoid-induced bone loss and hypercalciuria in mice: A comparative study with alendronate.

doi: 10.3892/ijmm.2016.2622

Figure Lengend Snippet: Figure 4. Expression of calcium transport proteins in the duodenum. (A and B) The protein expression of calcium transporters [transient receptor potential vanilloid (TRPV)5, TRPV6, calbindin-D9k (CaBP-9k)] was mea sured in the duodenum. Values are expressed as the means ± SEM, n=8 in each group. *P<0.05 versus vehicle group; #P<0.05 versus dexametha sone (DXM) group. Vehicle, vehicle group (n=8); DXM group, mice were injected intramuscularly with 5 mg/kg body weight DXM three times/week for 12 weeks (n=8); ALN group, mice received alendronate orally at a dose of 0.1 mg/kg/day in combination with DXM for 12 weeks (n=8); pomegranate seed (PS) group, mice received daily drinks of aqueous extract of pomegra nate seed (AE-PS) in combination with DXM for 12 weeks (n=8).

Article Snippet: After saturation with 5% (w/v) non-fat dry milk in Tris-buffered saline (TBS) and 0.1% (w/v) Tween-20 (TBST), the membranes were incubated with the following antibodies: transient receptor potential vanilloid (TRPV)5 (sc-398345; 1:500), TRPV6 (sc-28763; 1:500), osteoprotegerin (OPG; sc-8468; 1:1,000) and receptor activator of nuclear factor-κB ligand (RANKL; sc-7628; 1:2,000) , CaSR (sc-32181; 1:500), calbindin-D9k (CaBP-9k; sc-367381; 1:500), plasma membrane Ca2+-ATPase1 (PMCA1; sc-16488; 1:500) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), overnight at 4 ̊C.

Techniques: Expressing, Injection

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