Journal: Toxicology

Article Title: Differential Fmo3 Gene Expression in Various Liver Injury Models Involving Hepatic Oxidative Stress in Mice

doi: 10.1016/j.tox.2014.08.013

Figure Lengend Snippet: Livers were collected from mice (n=6) sacrificed at respective time-points (2,4,8,12,24 & 48h for ANIT; 24 & 48h for CCl4; 6 & 24h for AlOH; and 10 d for BDL). RNA was isolated and cDNA was made using a commercial MMLV-RT kit. The cDNA samples were analyzed by quantitative RT-PCR using Fmo3 mouse-specific primers. Gene expression was normalized to the housekeeping gene β-actin. Fmo3 mRNA expression is presented as mean Fold Change ± SE. One-way ANOVA, t-test or two-way ANOVA was performed, appropriately, followed by the Dunnett's posttest for One-way ANOVA and the Bonferroni posttest for two-way ANOVA. Asterisks (*) represent a statistical difference (p < 0.05) between vehicle-treated and hepatotoxicant-treated or BDL group.

Article Snippet: Membranes were blocked with 5 % non-fat powdered milk in tris buffered saline containing 0.05% tween-20(TBS-T) for 8 h. A rabbit anti-mouse Fmo3 primary antibody (GenScript USA Inc., NJ) (1:5000) was used to detect Fmo3 with β-actin as a loading control.

Techniques: Isolation, Quantitative RT-PCR, Expressing

Journal: Toxicology

Article Title: Differential Fmo3 Gene Expression in Various Liver Injury Models Involving Hepatic Oxidative Stress in Mice

doi: 10.1016/j.tox.2014.08.013

Figure Lengend Snippet: Western immunoblots for Fmo3 were performed using liver microsomes from control and hepatotoxicant-treated or BDL mice. A custom-made rabbit anti-mouse Fmo3 primary antibody, described in Materials and Methods was used to detect Fmo3. Fmo3 protein levels were normalized to β-actin loading control. Microsomal proteins isolated from naïve female mouse liver were used as a positive control (indicated by “+” sign). The data are presented as blots and as mean Fmo3 protein expression (Fold Change) ± SE. One-way ANOVA, t-test or two-way ANOVA was performed, appropriately, followed by the Dunnett's posttest for One-way ANOVA and the Bonferroni posttest for two-way ANOVA. Asterisks (*) represent a statistical difference (p < 0.05) between vehicle-treated and hepatotoxicant-treated or BDL group.

Article Snippet: Membranes were blocked with 5 % non-fat powdered milk in tris buffered saline containing 0.05% tween-20(TBS-T) for 8 h. A rabbit anti-mouse Fmo3 primary antibody (GenScript USA Inc., NJ) (1:5000) was used to detect Fmo3 with β-actin as a loading control.

Techniques: Western Blot, Isolation, Positive Control, Expressing

Journal: Toxicology

Article Title: Differential Fmo3 Gene Expression in Various Liver Injury Models Involving Hepatic Oxidative Stress in Mice

doi: 10.1016/j.tox.2014.08.013

Figure Lengend Snippet: Plasma and livers were collected from mice 72 h following APAP (400 mg/kg) or vehicle treatment. (A) The data are presented as mean plasma ALT (IU/L) ± SE. (B) RNA was isolated from livers andcDNA samples were analyzed by quantitative RT-PCR using Fmo3 mouse-specific primers. Gene expression was normalized to the housekeeping gene β-actin. Fmo3 mRNA expression are presented as mean Fold Change ± SE. Oneway ANOVA was performed followed by the Dunnett's post-test. Asterisks (*) represent a statistical difference (p < 0.05) between vehicle-treated group and APAP-treated group and hash (#) represent a statistical difference (p<0.05) compared with APAP-treated wild-type mice.

Article Snippet: Membranes were blocked with 5 % non-fat powdered milk in tris buffered saline containing 0.05% tween-20(TBS-T) for 8 h. A rabbit anti-mouse Fmo3 primary antibody (GenScript USA Inc., NJ) (1:5000) was used to detect Fmo3 with β-actin as a loading control.

Techniques: Isolation, Quantitative RT-PCR, Expressing

Journal: Toxicology

Article Title: Differential Fmo3 Gene Expression in Various Liver Injury Models Involving Hepatic Oxidative Stress in Mice

doi: 10.1016/j.tox.2014.08.013

Figure Lengend Snippet: After overnight fasting, groups of wild-type and Nrf2 knockout mice received a single dose of 400 mg/kg APAP or vehicle. Livers were collected 72 h following APAP or vehicle treatments. Western blot for Fmo3 was performed using liver microsomes from control and APAP-treated mice. Equal protein loading (10 μg protein/lane) was confirmed by detection of β-actin. Microsomal proteins isolated from naïve female mouse liver were used as a positive control indicated by “+” sign. The data are presented as blots and as mean Fmo3 protein expression (Fold Change) ± SE (A). FMO activity was measured in liver microsomes from control and APAP-treated mice using methimazole as substrate. Data are presented as mean Specific Activity (μM/min/mg) ± SE (B). Asterisks (*) represent a statistical difference (p < 0.05) between vehicle-treated group and APAP-treated group and hash (#) represent a statistical difference (p<0.05) compared with APAP-treated wild-type mice.

Article Snippet: Membranes were blocked with 5 % non-fat powdered milk in tris buffered saline containing 0.05% tween-20(TBS-T) for 8 h. A rabbit anti-mouse Fmo3 primary antibody (GenScript USA Inc., NJ) (1:5000) was used to detect Fmo3 with β-actin as a loading control.

Techniques: Knock-Out, Western Blot, Isolation, Positive Control, Expressing, Activity Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Directly auto-transplanted mesenchymal stem cells induce bone formation in a ceramic bone substitute in an ectopic sheep model

doi: 10.1111/j.1582-4934.2010.01131.x

Figure Lengend Snippet: Sheep MSC were characterized using FACS and RT-PCR analysis. (A) With RT-PCR analysis CD29, CD44 and CD166 expression of MSC could be proofed on mRNA level. As indicated by increased CD45 expression, ratio of hematopoietic cells was higher in directly auto-transplanted MSC as compared to expanded MSC. (B–D) FACS analysis revealed sheep MSC to express CD29, CD44 and CD166. Expanded MSC (B) were negative for the hematopoietic markers CD31 and CD45. Directly auto-transplanted cells (C) had a different expression pattern than expanded MSC. The directly auto-transplanted MSC had a weaker CD29 and CD166 but a stronger CD45 expression. Mean fluorescent indices are shown in (D).

Article Snippet: CD31 staining: After antigen retrieval with pH 6 solution (Target Retrieval Solution; Dako Cytomation) in a pressure cooker for 10 min. (Pascal; Dako Cytomation) peroxidase block (CSAII-System; Dako Cytomation) was applied for 15 min., followed by incubation with 10% goat serum (PromoCell GmbH) in PBS (PBS-Dulbecco 1×, Biochrom AG) for 30 min. and protein block with the CSA II-System for 30 min. Then sections were incubated with the primary monoclonal mouse anti-ovine antibody CD31 (Anti-CD31/PECAM-1, MorphoSys UK Ltd., Kidlington, Oxford, UK) at 1:100 diluted in 10% goat serum in PBS for 1 hr.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Directly auto-transplanted mesenchymal stem cells induce bone formation in a ceramic bone substitute in an ectopic sheep model

doi: 10.1111/j.1582-4934.2010.01131.x

Figure Lengend Snippet: For determination of the cell type which is qualified best for bone tissue engineering purposes, different groups (expanded versus directly auto-transplanted MSC, groups 8–10) were investigated. In both groups cells were DiI labelled prior to implantation and implanted subcutaneously with or without BMP-2. (A–C) Expanded MSC (A), directly auto-transplanted MSC (B), BMP-2 in combination with directly auto-transplanted MSC (C). DiI-labelled MSC (red) could be found close to β-TCP/HA granules contributing to the newly formed bone parts. In the explants with directly auto-transplanted MSC a higher section of the DiI-labelled cells were found in the connective tissue parts of the constructs compared to the explants with expanded MSC or directly auto-transplanted MSC with BMP-2. (D–F) Sections of constructs of the groups with expanded MSC (D), directly auto-transplanted MSC (E), BMP-2 in combination with directly auto-transplanted MSC (F) were evaluated for vascularization. The constructs in all three groups are well vascularized as shown by CD31 immunohistochemistry (green). Nuclei are counterstained with DAPI (blue).

Article Snippet: CD31 staining: After antigen retrieval with pH 6 solution (Target Retrieval Solution; Dako Cytomation) in a pressure cooker for 10 min. (Pascal; Dako Cytomation) peroxidase block (CSAII-System; Dako Cytomation) was applied for 15 min., followed by incubation with 10% goat serum (PromoCell GmbH) in PBS (PBS-Dulbecco 1×, Biochrom AG) for 30 min. and protein block with the CSA II-System for 30 min. Then sections were incubated with the primary monoclonal mouse anti-ovine antibody CD31 (Anti-CD31/PECAM-1, MorphoSys UK Ltd., Kidlington, Oxford, UK) at 1:100 diluted in 10% goat serum in PBS for 1 hr.

Techniques: Construct, Immunohistochemistry

Journal:

Article Title: Retinoid Metabolism and ALDH1A2 (RALDH2) Expression are Altered in the Transgenic Adenocarcinoma Mouse Prostate Model

doi: 10.1016/j.bcp.2009.06.022

Figure Lengend Snippet: Primer Sequences Used for Quantitative RT-PCR

Article Snippet: 2.5 ALDH1A2 Polyclonal Antibody Generation A rabbit anti-mouse ALDH1A2 polyclonal antibody was generated by contract with Alpha Diagnostics International (San Antonio, TX).

Techniques: Sequencing

Journal:

Article Title: Retinoid Metabolism and ALDH1A2 (RALDH2) Expression are Altered in the Transgenic Adenocarcinoma Mouse Prostate Model

doi: 10.1016/j.bcp.2009.06.022

Figure Lengend Snippet: A, Total RNA was extracted and reverse-transcribed from prostate lobes microdissected from three nontransgenic mice at 18, 24, and 36 weeks of age. The bars indicate the average transcript levels of ALDH1A1, ALDH1A2, and ALDH1A3 in prostate tissue from age-matched nontransgenic controls (WT, black bars) and TRAMP (white bars) normalized to 36B4 mRNA levels in each sample. B, Relative mRNA levels of RARβ2, CYP26A1, and LRAT measured by quantitative RT-PCR in WT (black bars) and TRAMP mice (white bars) at 36 weeks of age. All samples were normalized to 36B4. VP, ventral prostate; LP, lateral prostate; DP, dorsal prostate; and AP, anterior prostate. Relative expression was calculated using the Bio-Rad Genex software, where all prostate lobes from a given age group were processed independently. Error bars= standard error. Comparisons for statistical analysis were made for each lobe between the Wt and the TRAMP mice at each of the time points. *, p ≤ 0.05 as determined by two-tailed Student's t test, comparing TRAMP mice to their age-matched littermate controls. (TRAMP = TRAMP+).

Article Snippet: 2.5 ALDH1A2 Polyclonal Antibody Generation A rabbit anti-mouse ALDH1A2 polyclonal antibody was generated by contract with Alpha Diagnostics International (San Antonio, TX).

Techniques: Reverse Transcription, Quantitative RT-PCR, Expressing, Software, Two Tailed Test

Journal:

Article Title: Retinoid Metabolism and ALDH1A2 (RALDH2) Expression are Altered in the Transgenic Adenocarcinoma Mouse Prostate Model

doi: 10.1016/j.bcp.2009.06.022

Figure Lengend Snippet: A, Wild type mouse embryo at E12/13 (WT). ALDH1A2 staining located in the metanephros. B, ALDH1A2-/- embryo at E12/13. No ALDH1A2 staining was observed in the metanephros, confirming antibody specificity. E, Wild type mouse testis at 36 weeks of age. ALDH1A2 staining located in the germ cells but not in the spermatagonia. G, Wild type mouse kidney at 36 weeks of age. ALDH1A2 staining was observed in the tubular cells, but not in the glomeruli. C-D and G-H, Negative control incubated with preimmune serum instead of primary antibody. A-D, 200× magnification. E-H, 400× magnification.

Article Snippet: 2.5 ALDH1A2 Polyclonal Antibody Generation A rabbit anti-mouse ALDH1A2 polyclonal antibody was generated by contract with Alpha Diagnostics International (San Antonio, TX).

Techniques: Staining, Negative Control, Incubation

Journal:

Article Title: Retinoid Metabolism and ALDH1A2 (RALDH2) Expression are Altered in the Transgenic Adenocarcinoma Mouse Prostate Model

doi: 10.1016/j.bcp.2009.06.022

Figure Lengend Snippet: Tissue extracts were obtained from microdissected dorsal prostate lobes (30 μg protein loaded/lane) from nontransgenic (labeled as WT) littermate controls and TRAMP mice at 18, 24, and 36 weeks of age (3 mice per condition). A, Western blot analysis performed with the polyclonal rabbit anti-mouse ALDH1A2 antibody. B, Western blot analysis performed with the polyclonal rabbit anti-human S100A4 antibody. For a loading control, these blots were stripped and reblotted with a polyclonal goat anti-human actin antibody. Positive controls: ALDH1A2-wild type testis extract (15 μg protein loaded/lane); S100A4-PC3 cell extract (30 μg protein loaded/lane). This experiment was performed three times with similar results; one blot is shown. The upper arrow at the right in panel A shows ALDH1A2; the lower arrow points to the non-specific protein band at ∼30 kd. C, Densitometric quantitation of similar ALDH1A2 and S100A4 Western Blots. Band density was measured using the Fluor Chem 8800 software (Alpha Innotech) for the bands from ALDH1A2 (left) and S100A4 (right). Western blots normalized to actin for all prostate samples. Error bars = standard error. *, p ≤ 0.05 as determined by two-tailed Student's t test. TRAMP mice were compared to age-matched littermate nontransgenic controls (WT). DP, dorsal prostate.

Article Snippet: 2.5 ALDH1A2 Polyclonal Antibody Generation A rabbit anti-mouse ALDH1A2 polyclonal antibody was generated by contract with Alpha Diagnostics International (San Antonio, TX).

Techniques: Labeling, Western Blot, Control, Quantitation Assay, Software, Two Tailed Test

Journal:

Article Title: Retinoid Metabolism and ALDH1A2 (RALDH2) Expression are Altered in the Transgenic Adenocarcinoma Mouse Prostate Model

doi: 10.1016/j.bcp.2009.06.022

Figure Lengend Snippet: All tissue sections were stained with hematoxylin (blue). A, C, E, and G, ALDH1A2 staining. B, D, F, and H, S100A4 staining. A-H, 600× magnification. Dorsal lobe (A) and lateral lobe (C) from an 18 week old nontransgenic mouse. Strong nuclear and cytoplasmic ALDH1A2 staining (brown stain) representative of normal prostate epithelial cells in all lobes. S100A4 (brown stain) staining in fibroblast cells surrounding a normal prostate gland in dorsal lobe (B) and lateral lobe (D). E, dorsal prostate tumor tissue in a 36 week old TRAMP mouse, showing weak cytoplasmic ALDH1A2 staining in prostate cancer cells with little to no stain in the nuclei. There is no ALDH1A2 staining in the stroma of dorsal prostate tissue in the TRAMP mouse. F, S100A4 staining in the stroma of TRAMP mice and among prostate cancer cells within the gland. G, lateral prostate tumor tissue in a 36 week old TRAMP mouse. The ALDH1A2 staining pattern in the prostate cancer cells is similar to E. Positive staining of ALDH1A2 in the lateral prostate stroma, which is unique to this lobe. F, S100A4 staining in the prostate cancer and lateral prostate stroma. I-J, Negative controls incubated with preimmune serum instead of primary antibody. I, prostate tumor tissue from dorsal prostate of 30 week old TRAMP mouse, 200× magnification. J, prostate tumor tissue from lateral prostate of 30 week old TRAMP mouse, 200× magnification. K, ALDH1A2 staining of prostate tumor tissue from 30 week old TRAMP mouse, 200× magnification. L, adjacent section of ALDH1A2 staining of prostate tumor tissue from 30 week old TRAMP mouse. Tumor tissue section was simultaneously incubated with the 20× peptides to which the antibody was generated. (Arrows indicate areas of sections discussed in the Results section).

Article Snippet: 2.5 ALDH1A2 Polyclonal Antibody Generation A rabbit anti-mouse ALDH1A2 polyclonal antibody was generated by contract with Alpha Diagnostics International (San Antonio, TX).

Techniques: Staining, Incubation, Generated

Journal:

Article Title: Retinoid Metabolism and ALDH1A2 (RALDH2) Expression are Altered in the Transgenic Adenocarcinoma Mouse Prostate Model

doi: 10.1016/j.bcp.2009.06.022

Figure Lengend Snippet: A-J, representative slides of a human prostate tumor tissue. A-B and E-F, ALDH1A2 staining at 200× and 600× (boxed areas of A and E) magnification. B and F, ALDH1A2 staining located in the cytoplasmic compartment of basal and luminal cells in a normal prostate gland with weak staining in adjacent cancer cells. C-D and G-H, S100A4 staining at 200× and 600× (boxed areas of C and G) magnification. S100A4 staining is seen in benign glands, as well as in surrounding stroma. I-J, Negative controls, prostate specimens incubated with preimmune serum instead of primary antibody, 200× magnification. (Arrows indicate areas on sections discussed in the Results section).

Article Snippet: 2.5 ALDH1A2 Polyclonal Antibody Generation A rabbit anti-mouse ALDH1A2 polyclonal antibody was generated by contract with Alpha Diagnostics International (San Antonio, TX).

Techniques: Staining, Incubation