secondary antibody solution  (Vector Laboratories)

Journal: Biochemistry and Biophysics Reports

Article Title: Oxysterol-binding protein-related protein 6 regulates neuronal morphology and migration of cerebellar granule cells during cerebellar development in vivo

doi: 10.1016/j.bbrep.2026.102585

Figure Lengend Snippet: ORP6 RNAi decreases cell motility of primary cultured cerebellar granule cells (CGCs). Cell-tracking images of primary cultured CGCs transfected with control RNA (A) or ORP6 RNAi (B), stained with Hoechst. (C) The accumulated distance of primary cultured CGCs transfected with control or ORP6 RNAi is automatically analyzed by PerkinElmer Harmony 4.9 Image Analysis Software. All colored arrows indicate the distance and direction of cell movement. Data are collected from five independent cell culture preparations, and the accumulated distance of each two groups is shown as the mean ± SE. Statistical analysis is performed using Welch's t -test. A P value less than 0.05 is considered statistically significant. Bars, 50 μm.

Article Snippet: Neuro-2A cells, primary cultured CGCs, and cerebellar sections were incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies at 37 °C for 1 h, as described in , Cells and cerebellar sections were then washed four times with PBS and incubated with Hoechst stain (346-07951, DOJINDO, Kumamoto, Japan) in PBS at RT for 10 min. After washing with PBS, the cerebellar sections were mounted with CC/Mount (K002, Diagnostic Biosystems, Pleasanton, CA, USA).

Techniques: Cell Culture, Cell Tracking Assay, Transfection, Control, Staining, Software

Journal: Biochemistry and Biophysics Reports

Article Title: Oxysterol-binding protein-related protein 6 regulates neuronal morphology and migration of cerebellar granule cells during cerebellar development in vivo

doi: 10.1016/j.bbrep.2026.102585

Figure Lengend Snippet: ORP6 int impaired the migration of cerebellar granule cells (CGCs) in the developing cerebellum. (A) Experimental design of gene transfection into P7 mice cerebellum by in vivo electroporation and tissue collection. Sagittal section of P9 cerebellum transfected with pCAGGS-AcGFP-C (B–D) or pCAGGS-AcGFP-C-ORP6 int (E–G), and immunostained with anti-calbindin antibody (C and F). The cerebellar laminar structure is identified as follows: the calbindin-positive Purkinje cell layer (PCL) and molecular layer (ML), which lies superficial to the PCL and contains sparsely Hoechst-stained nuclei. The external granular layer is the outermost layer of the ML, a region with dense Hoechst-stained nuclei, and the internal granular layer located beneath the calbindin-positive PCL. Arrows indicate distribution of CGCs expressing pCAGGS-AcGFP-C or pCAGGS-AcGFP-C-ORP6 int. Ratio of cells transfected with pCAGGS-AcGFP-C (H) or pCAGGS-AcGFP-C-ORP6 int (I) in each layer to total cells. Data are collected from four animals, and the cell number of each two groups is shown as the mean ± SE. Statistical analysis is performed using Welch's t -test. A P value less than 0.05 is considered statistically significant. Bars, 50 μm.

Article Snippet: Neuro-2A cells, primary cultured CGCs, and cerebellar sections were incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies at 37 °C for 1 h, as described in , Cells and cerebellar sections were then washed four times with PBS and incubated with Hoechst stain (346-07951, DOJINDO, Kumamoto, Japan) in PBS at RT for 10 min. After washing with PBS, the cerebellar sections were mounted with CC/Mount (K002, Diagnostic Biosystems, Pleasanton, CA, USA).

Techniques: Migration, Transfection, In Vivo, Electroporation, Staining, Expressing

Journal: Biomedical Reports

Article Title: Peroxiredoxin 4 suppresses ferroptosis in esophageal squamous cell carcinoma by activating the phosphoinositide 3-kinase  signaling pathway

doi: 10.3892/br.2026.2133

Figure Lengend Snippet: PRDX4 is an important regulator of ferroptosis in ESCC cells. (A) Determination of MDA, LPO and GSH contents in KYSE270 cells after transfection with PRDX4 siRNA. (B) Western blot analysis of the protein levels of GPX4, SLC7A11 and PTGS2 in KYSE270 cells transfected with PRDX4 siRNA. (C) The relative protein levels of GPX4, SLC7A11 and PTGS2 in KYSE270 cells transfected with PRDX4 siRNA. (D) Determination of MDA, LPO and GSH contents in KYSE30 cells after transfection with pcDNA3.1-PRDX4. (E) Western blot analysis of the protein levels of GPX4, SLC7A11 and PTGS2 in KYSE30 cells transfected with pcDNA3.1-PRDX4. (F) The relative protein levels of GPX4, SLC7A11 and PTGS2 in KYSE30 cells transfected with pcDNA3.1-PRDX4. (G) Detection of the levels of MDA, LPO and GSH in the control group, PRDX4 siRNA group and PRDX4 siRNA plus Fer-1 group in KYSE270 cells. (H) Western blot analysis of the protein expression levels of GPX4, SLC7A11 and PTGS2 in the control group, PRDX4 siRNA group and PRDX4 siRNA plus Fer-1 group in KYSE270 cells. (I) The relative protein levels of GPX4, SLC7A11 and PTGS2 in the control group, PRDX4 siRNA group and PRDX4 siRNA plus Fer-1 group in KYSE270 cells. (J) Detection of the levels of MDA, LPO and GSH in the pcDNA3.1 group, pcDNA3.1-PRDX4 group and pcDNA3.1-PRDX4 plus erastin group in KYSE30 cells. (K) Western blot analysis of the protein expression levels of GPX4, SLC7A11 and PTGS2 in the pcDNA3.1 group, pcDNA3.1-PRDX4 group and pcDNA3.1-PRDX4 plus erastin group in KYSE30 cells. (L) The relative protein levels of GPX4, SLC7A11 and PTGS2 in the pcDNA3.1 group, pcDNA3.1-PRDX4 group and pcDNA3.1-PRDX4 plus erastin group in KYSE30 cells. ** P<0.01, *** P<0.001 and **** P<0.0001, indicate statistical significance. PRDX4, peroxiredoxin 4; ESCC, esophageal squamous cell carcinoma; siRNA, small interfering RNA; MDA, malondialdehyde; LPO, lipid peroxidation; GSH, glutathione; GPX4, glutathione peroxidase 4; SLC7A11, solute carrier family 7 member 11; PTGS2, prostaglandin-endoperoxide synthase 2; Fer-1, ferrostatin-1; ns, not significant.

Article Snippet: The primary antibodies were as follows: Anti-PRDX4 (1:2,000 dilution; cat. no. 10703-1-AP), anti-E-cadherin (1:20,000 dilution; cat. no. 20874-1-AP), anti-N-cadherin (1:2,000 dilution; cat. no. 22018-1-AP), anti-Vimentin (1:20,000 dilution; cat. no. 10366-1-AP), and anti-GPX4 (1:1,000 dilution; cat. no. 30388-1-AP; all from Proteintech Group, Inc.), anti-solute carrier family 7 member 11 (SLC7A11; 1:1,000 dilution; cat. no. DF12509; Affinity Biosciences, Ltd.), anti-prostaglandin-endoperoxide synthase 2 (PTGS2; 1:1,000 dilution; cat. no. 27308-1-AP; Proteintech Group, Inc.), anti-p-PI3K (1:1,000 dilution; cat. no. AP0427; ABclonal Biotech Co., Ltd.), anti-PI3K (1:1,000 dilution; cat. no. A19742; ABclonal Biotech Co., Ltd.), anti-p-AKT (1:2,000 dilution; cat. no. 28731-1-AP; Proteintech Group, Inc.), anti-AKT (1:2,000 dilution; cat. no. 10176-2-AP; Proteintech Group, Inc.) and anti-β-actin (1:2,000 dilution; cat. no. 20536-1-AP; Proteintech Group, Inc.).

Techniques: Transfection, Western Blot, Control, Expressing, Small Interfering RNA

Journal: Neuropsychopharmacology Reports

Article Title: Distinct Effects of Nonselective Rho‐Kinase Inhibitor Fasudil and Selective Rho‐Kinase 2 Inhibitor KD025 on Serotonin and Dopamine Release in the Nucleus Accumbens of Mice

doi: 10.1002/npr2.70124

Figure Lengend Snippet: Representative image of the guide cannula trace in the NAc. Blue signals represent Hoechst 33342. White dashed line represents the outline of the guide cannula (scale bar indicates 500 μm). NAc, nucleus accumbens.

Article Snippet: After washing in PBS, the sections were incubated with the secondary antibody with Hoechst 33342 (346–07951, 1:2000, Dojindo, Kumamoto, Japan) at 25°C for 1 h. The secondary antibodies were donkey anti‐rabbit Alexa Fluor 488 (Cat# A32790TR, RRID:AB_2866495, 1:2000 dilution; Thermo Fisher Scientific, Waltham, MA, USA), donkey anti‐rat Alexa Fluor 568 (Cat# A78946, RRID:AB_2910653, 1:1000 dilution; Thermo Fisher Scientific), and donkey anti‐goat Alexa Fluor 647 (Cat# A32849TR, RRID:AB_2866498, 1:1000 dilution; Thermo Fisher Scientific).

Techniques:

Journal: Neuropsychopharmacology Reports

Article Title: Distinct Effects of Nonselective Rho‐Kinase Inhibitor Fasudil and Selective Rho‐Kinase 2 Inhibitor KD025 on Serotonin and Dopamine Release in the Nucleus Accumbens of Mice

doi: 10.1002/npr2.70124

Figure Lengend Snippet: Co‐expression of Rho‐kinases 1 and 2 with SERT or DAT. A: Representative immunoreactivity images of Rho‐kinase 1 (green), SERT (magenta), DAT (red), and Hoechst 33342 (blue) in the NAc (scale bar indicates 10 μm). B: Percentage of Rho‐kinase 1 and the SERT or DAT double‐positive area to the SERT or DAT‐positive area in the NAc. C: Representative immunoreactivity images of Rho‐kinase 2 (green), SERT (magenta), DAT (red), and Hoechst 33342 (blue) in the NAc (scale bar indicates 10 μm). D: Percentage of Rho‐kinase 2 and SERT or DAT double‐positive area relative to the SERT or DAT‐positive area in the NAc. Data represent the mean + SEM ( n = 5). DAT, dopamine transporter; SERT, serotonin transporter; NAc, nucleus accumbens.

Article Snippet: After washing in PBS, the sections were incubated with the secondary antibody with Hoechst 33342 (346–07951, 1:2000, Dojindo, Kumamoto, Japan) at 25°C for 1 h. The secondary antibodies were donkey anti‐rabbit Alexa Fluor 488 (Cat# A32790TR, RRID:AB_2866495, 1:2000 dilution; Thermo Fisher Scientific, Waltham, MA, USA), donkey anti‐rat Alexa Fluor 568 (Cat# A78946, RRID:AB_2910653, 1:1000 dilution; Thermo Fisher Scientific), and donkey anti‐goat Alexa Fluor 647 (Cat# A32849TR, RRID:AB_2866498, 1:1000 dilution; Thermo Fisher Scientific).

Techniques: Expressing