anti class iii betatubulin tubb3 antibody  (R&D Systems)

Journal: Brain Communications

Article Title: Survival motor neuron protein-independent amelioration of spinal muscular atrophy by pharmacological inhibition of c-Jun-NH 2 terminal kinase

doi: 10.1093/braincomms/fcag111

Figure Lengend Snippet: The effect of JNK inhibitors on preventing the degeneration of SMN-deficient cultured primary neurons and spinal cord motor neurons derived from SMA mice. Cultured primary neurons (CGN) were transfected with siRNA (siSMN) 100 nM and incubated for 24 h before treatment with inhibitors (1.0 μM). Neurons were fixed after 24 h treatment of inhibitor with 4% PFA and stained with antibodies to neuron-specific β-tubulin class III (red) and p-c-Jun (green) and IF examined by confocal laser scanning microscopy. Degeneration of neurons caused by SMN knockdown (siSMN) is shown by axonal degeneration compared with control neurons stained with tubulin (red). Nuclei were stained with DAPI (blue). ( A ) Treatment with JNKII, a negative control for JNK inhibition, did not cause decrease in p-c-Jun (green) levels (siSMN + JNKII) and did not reduce the degeneration of neurons compared with SMN-deficient neurons (siSMN). Treatments with ( B ) SP600125, ( C ) AS601245, and ( D ) SR12519 show JNK inhibition causes a decrease in p-c-Jun (green) levels (siSMN + SP600125) compared with SMN-deficient neurons (siSMN) and results in a reduction in the degeneration of neurons (red) showing protection of SMN-deficient neurons by pharmacological JNK inhibition. ( E ) Pharmacological inhibition of JNK prevents degeneration and improves the growth of spinal cord motor neurons derived from SMA mice. Primary spinal cord neurons were cultured from 7-day-old normal and SMA mice littermates. Neurons were differentiated in vitro for 14 days, treated with JNK inhibitors (1.0 μM) for 6 days with a change of medium supplemented with fresh inhibitors every 48 h and fixed with 4% PFA. Fixed neurons were stained with antibodies against neuron-specific β-tubulin-III (red), and p-c-Jun (green), and IF was examined by confocal microscopy. Neurons from normal mice show healthy motor neurons with very low staining for p-c-Jun (green). Neurons from SMA mice (SMA + DMSO) show signs of degeneration (arrows) and high staining for p-c-Jun (green) compared with neurons from normal mice. Treatments with JNK inhibitors, SP600125, AS601245, and SR12519 reduces JNK and causes a decrease in p-c-Jun (green) levels (SMA + SP600125 or AS601245 or SR12519) compared with SMA neurons treated with DMSO (SMA + DMSO), leading to protection and improved growth of SMA spinal cord motor neurons by pharmacological JNK inhibition. Scale bar is 50 μm.

Article Snippet: PFA fixed primary neurons were washed with PBS, permeabilized with 0.1% Triton-X100 for 5 min, washed 3 × 5 min with PBS with 0.2% Tween-20 (PBS-T), blocked with 3% BSA in PBS-T for 30 min and double labelled using sequential incubation (1 h each) with primary antibodies phospho-c-Jun (p-c-Jun) (Ser63) #9261, (Cell Signaling) and followed by secondary antibody Alexa 488-conjugated IgG (green), washed 3 × 5 min with PBS-T and incubated with second primary antibody, anti-β-tubulin class-III neuron-specific antibody anti-β-tubulin class-III neuron-specific, (TUJ1, R&D systems), washed 3 × 5 min with PBS-T followed by incubation with Alexa 594-conjugated IgG (red) anti-mouse or anti-rabbit IgG secondary antibody.

Techniques: Cell Culture, Derivative Assay, Transfection, Incubation, Staining, Confocal Laser Scanning Microscopy, Knockdown, Control, Negative Control, Inhibition, In Vitro, Confocal Microscopy

Journal: Brain Communications

Article Title: Survival motor neuron protein-independent amelioration of spinal muscular atrophy by pharmacological inhibition of c-Jun-NH 2 terminal kinase

doi: 10.1093/braincomms/fcag111

Figure Lengend Snippet: The effect of JNK inhibitors on preventing the degeneration of SMN-deficient cultured primary neurons and spinal cord motor neurons derived from SMA mice. Cultured primary neurons (CGN) were transfected with siRNA (siSMN) 100 nM and incubated for 24 h before treatment with inhibitors (1.0 μM). Neurons were fixed after 24 h treatment of inhibitor with 4% PFA and stained with antibodies to neuron-specific β-tubulin class III (red) and p-c-Jun (green) and IF examined by confocal laser scanning microscopy. Degeneration of neurons caused by SMN knockdown (siSMN) is shown by axonal degeneration compared with control neurons stained with tubulin (red). Nuclei were stained with DAPI (blue). ( A ) Treatment with JNKII, a negative control for JNK inhibition, did not cause decrease in p-c-Jun (green) levels (siSMN + JNKII) and did not reduce the degeneration of neurons compared with SMN-deficient neurons (siSMN). Treatments with ( B ) SP600125, ( C ) AS601245, and ( D ) SR12519 show JNK inhibition causes a decrease in p-c-Jun (green) levels (siSMN + SP600125) compared with SMN-deficient neurons (siSMN) and results in a reduction in the degeneration of neurons (red) showing protection of SMN-deficient neurons by pharmacological JNK inhibition. ( E ) Pharmacological inhibition of JNK prevents degeneration and improves the growth of spinal cord motor neurons derived from SMA mice. Primary spinal cord neurons were cultured from 7-day-old normal and SMA mice littermates. Neurons were differentiated in vitro for 14 days, treated with JNK inhibitors (1.0 μM) for 6 days with a change of medium supplemented with fresh inhibitors every 48 h and fixed with 4% PFA. Fixed neurons were stained with antibodies against neuron-specific β-tubulin-III (red), and p-c-Jun (green), and IF was examined by confocal microscopy. Neurons from normal mice show healthy motor neurons with very low staining for p-c-Jun (green). Neurons from SMA mice (SMA + DMSO) show signs of degeneration (arrows) and high staining for p-c-Jun (green) compared with neurons from normal mice. Treatments with JNK inhibitors, SP600125, AS601245, and SR12519 reduces JNK and causes a decrease in p-c-Jun (green) levels (SMA + SP600125 or AS601245 or SR12519) compared with SMA neurons treated with DMSO (SMA + DMSO), leading to protection and improved growth of SMA spinal cord motor neurons by pharmacological JNK inhibition. Scale bar is 50 μm.

Article Snippet: PFA fixed primary neurons were washed with PBS, permeabilized with 0.1% Triton-X100 for 5 min, washed 3 × 5 min with PBS with 0.2% Tween-20 (PBS-T), blocked with 3% BSA in PBS-T for 30 min and double labelled using sequential incubation (1 h each) with primary antibodies phospho-c-Jun (p-c-Jun) (Ser63) #9261, (Cell Signaling) and followed by secondary antibody Alexa 488-conjugated IgG (green), washed 3 × 5 min with PBS-T and incubated with second primary antibody, anti-β-tubulin class-III neuron-specific antibody anti-β-tubulin class-III neuron-specific, (TUJ1, R&D systems), washed 3 × 5 min with PBS-T followed by incubation with Alexa 594-conjugated IgG (red) anti-mouse or anti-rabbit IgG secondary antibody.

Techniques: Cell Culture, Derivative Assay, Transfection, Incubation, Staining, Confocal Laser Scanning Microscopy, Knockdown, Control, Negative Control, Inhibition, In Vitro, Confocal Microscopy

Journal: Cellular and Molecular Neurobiology

Article Title: Diverse Effects of Various Toll-Like Receptor 2 Ligands on Neuronal Activity and Cell Death

doi: 10.1007/s10571-025-01632-3

Figure Lengend Snippet: Expression of TLR2 in the cortical-derived neurons and other cells. Primary rat cortical cells were cultured for 2–16 days. ( a ) Representative images of primary cortical cells at DIV2, DIV9, DIV12, and DIV16. Representative images showing the signal from Hoechst 33342-stained nuclei in blue, beta III tubulin immunoreactivity in green, and TLR2 immunoreactivity in red in the cultured cortical cells, and merged image (bottom panel). (4 biological replicates in 1 technical replicate from 2 independent experiments) ( b ) Magnified images from ( a ) showing TLR2 in neurons. Scale bar: 100 μm. Cells were analyzed in 4 fields of view. Two independent experiments were performed

Article Snippet: Anti-class III beta-tubulin (TUBB3) antibody (R&D systems, MAB1195), a marker for neurons (Ballas et al. ), anti-TLR2 antibody (Thermo, MA532787 ), anti-glial fibrillary acidic protein (GFAP) antibody (abcam, ab68428), a marker for astrocytes (Shixing et al. ), anti-Iba1 antibody (CST, 17198T), a marker for microglia (Ni et al. ), and anti-Olig2 antibody (abcam, ab109186), a marker for oligodendrocytes (Xu et al. ), were used as primary antibodies (Wyczanska et al. ).

Techniques: Expressing, Derivative Assay, Cell Culture, Staining

Journal: Materials Today Bio

Article Title: Dorsal root ganglion-targeted analgesic delivery for effective relief of neuropathic pain

doi: 10.1016/j.mtbio.2025.102025

Figure Lengend Snippet: In vivo DRG-targeting ability of BK-LNPs in SNI and CINP. (A) t-distributed stochastic neighbor embedding (t-SNE) visualization of Adam8 mRNA expression patterns across DRG cellular subpopulations and (B) RNAscope imaging for Adam8 mRNA in the DRG neurons following SNI modeling. (C–D) Relative Adam8 mRNA expression level in the DRG on the 7th and 28th days following SNI modeling. (E) CLSM images of the bilateral DRGs ipsilateral and contralateral to SNI surgical site and (F) corresponding quantitative analysis of RhB fluorescence. Tuj1 was a neuronal marker. (G) Schematic illustration for the bilateral DRGs ipsilateral and contralateral to SNI surgical site. (H) Relative Adam8 mRNA expression level in the DRG as well as in the heart, liver, spleen, lung, and kidney on the 7th day following CINP modeling. (I) CLSM images of the DRG in CINP and (J) corresponding quantitative analysis of RhB fluorescence. For CLSM imaging, mice were intravenously injected with LNP/RhB and BK-LNPs/RhB and at 6 h post-injection, mice were euthanized for analysis. Data were expressed as mean ± SEM (n = 3 for C and D; n = 5 for F, H, and J). ∗∗ p < 0.01 and ∗∗∗ p < 0.001.

Article Snippet: The obtained DRG sections (14 μm in thickness) were blocked with 10 % BSA for 1 h and then sequentially incubated with neuronal class III β-tubulin (Tuj1) primary antibody (D71G9, rabbit, Cell Signaling Technology, #5568, 1:1000) overnight and its fluorescence-labeled secondary antibody (Alexa Fluor 488 anti-rabbit IgG, donkey, ThermoFisher, #A-21206, 1:500) for 2 h. The fluorescence of Tuj1 and RhB was captured on the SP8 CLSM using the corresponding filters to evaluate the targeting ability of BK-1361.

Techniques: In Vivo, Expressing, RNAscope, Imaging, Fluorescence, Marker, Injection