Journal: Parasite immunology

Article Title: SEA Alleviates Hepatic Ischaemia-Reperfusion Injury by Promoting M2 Macrophage Polarisation.

doi: 10.1111/pim.13061

Figure Lengend Snippet: FIGURE 4 | SEA pretreatment promoted the polarisation of M2 macrophages. Mice were subjected to 60 min of hepatic ischaemia, followed by 6 h of reperfusion. (A) Representatives of immunofluorescence staining of CD68 (n = 5 per group, five biological replicates from five individual animals; original magnification 100× and 200×, scale bar: 100 μm). (B) Representatives of immunofluorescence staining of CD163 (n = 5 per group, five biological replicates from five individual animals; original magnification 100× and 200×, scale bar: 100 μm). (C, D) The expression of M2 macrophage marker (CD206 and Arg1) in the liver was detected via qRT-PCR (n = 5 per group, five biological replicates from five individual animals). These results were obtained from at least three independent experiments. Values are presented as mean ± SEM. **p < 0.01, *p < 0.05, ns > 0.05.

Article Snippet: Then the sections were incubated with primary antibodies dissolved in 5% donkey serum solution containing 0.3% Triton at 4°C overnight (anti- MPO, Cat# ab208670, Abcam, 1:100; anti- CD68, Cat# ab53444, Abcam, 1:100; anti- CD163, Cat# 16646- 1- AP, Proteintech, 1:100).

Techniques: Immunofluorescence, Staining, Expressing, Marker, Quantitative RT-PCR

Journal:

Article Title: Enhancement of Recombinant Hemoglobin Production in Escherichia coli BL21(DE3) Containing the Plesiomonas shigelloides Heme Transport System ▿

doi: 10.1128/AEM.01291-08

Figure Lengend Snippet: Hemoglobin production in BL21(DE3) transformed with various plasmids. (A) Absorbance scans of live cultures of BL21(DE3) with and without hemoglobin or P. shigelloides heme transport genes. Solid line, E. coli containing hemoglobin and heme transport genes [(BL21(DE3)/pHB0.0, pHUG21]; dotted line, E. coli containing hemoglobin genes alone [BL21(DE3)/pHB0.0, pWSK29]; dashed line, E. coli containing heme transport genes alone [BL21(DE3)/pDSV1, pHUG21]. Values on the x axis are wavelengths in nanometers. (B) First derivative spectrum obtained from absorbance scans. Derivative values are shown beside each arrow. Solid line, BL21(DE3) containing heme transport and hemoglobin genes; dotted line, BL21(DE3) containing hemoglobin genes alone. (C) Immunoblot of soluble (S) and insoluble (I) fractions from 0.004 OD unit of cells at a wavelength of 700 nm. Lanes 1 and 4, BL21(DE3) containing heme transport genes alone; lanes 2 and 5, BL21(DE3) containing hemoglobin genes alone; lanes 3 and 6, BL21(DE3) containing hemoglobin and heme transport genes; lane 7, 0.45 μg of hemoglobin (Sigma Chemicals).

Article Snippet: Immunoblots using goat anti-human hemoglobin antibody (BiosPacific Fortron Bio Science, Inc.) and horseradish peroxidase-labeled rabbit anti-goat antibody (Bio-Rad) were performed on protein samples that had been electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and densitometry of autoradiograms was performed using the UVP AutoChemi system.

Techniques: Transformation Assay, Western Blot

Journal:

Article Title: Enhancement of Recombinant Hemoglobin Production in Escherichia coli BL21(DE3) Containing the Plesiomonas shigelloides Heme Transport System ▿

doi: 10.1128/AEM.01291-08

Figure Lengend Snippet: Hemoglobin production in BL21(DE3) containing the one-plasmid or two-plasmid system. (A) Genetic and partial restriction enzyme map of genes on pHB0.0hug. Genes are indicated with horizontal arrows, and promoters are indicated with vertical bent arrows. Restriction enzyme sites are as follows: B, BamHI; H, HindIII; N, NheI; S, SalI; and W, BsiWI. (B) Immunoblot of soluble (S) and insoluble (I) fractions. Lanes 1 and 3, BL21(DE3) containing the two-plasmid system (pHB0.0, pHUG21); lanes 2 and 4, BL21(DE3) containing the one-plasmid system (pHB0.0hug); lane 5, 0.45 μg of hemoglobin. (C) Densitometry analysis of immunoblots of soluble fractions. Data are from three independent sets of samples analyzed on the same immunoblot; soluble fractions from 0.004 OD unit of cells at a wavelength of 700 nm were loaded. Open circles, hemoglobin control; filled squares, BL21(DE3) containing the two-plasmid system; filled triangles, BL21(DE3) containing the one-plasmid system.

Article Snippet: Immunoblots using goat anti-human hemoglobin antibody (BiosPacific Fortron Bio Science, Inc.) and horseradish peroxidase-labeled rabbit anti-goat antibody (Bio-Rad) were performed on protein samples that had been electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and densitometry of autoradiograms was performed using the UVP AutoChemi system.

Techniques: Plasmid Preparation, Western Blot, Control

Journal:

Article Title: Enhancement of Recombinant Hemoglobin Production in Escherichia coli BL21(DE3) Containing the Plesiomonas shigelloides Heme Transport System ▿

doi: 10.1128/AEM.01291-08

Figure Lengend Snippet: Analysis of hemoglobin production in BL21(DE3) containing various plasmids

Article Snippet: Immunoblots using goat anti-human hemoglobin antibody (BiosPacific Fortron Bio Science, Inc.) and horseradish peroxidase-labeled rabbit anti-goat antibody (Bio-Rad) were performed on protein samples that had been electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and densitometry of autoradiograms was performed using the UVP AutoChemi system.

Techniques: Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Age-related decline of hemoglobin-α (Hb-α) with parallel increase in brain hypoxia in the intact male mouse brain. ( A ) Representative photomicrographs from male mouse hippocampal CA1, CA3 and cerebral cortex regions showing age-related decline in Hb-α staining and parallel increase in brain hypoxia as measured by pimonidazole (hypoxyprobe-1) staining. ( B ) Semi-quantitative intensity analysis of Hb-α and pimonidazole staining in hippocampal CA1 and CA3 regions and cerebral cortex of all male mice at different ages. ( C ) Correlation coefficient analysis between Hb-α and pimonidazole in hippocampal CA1, CA3 and cortex regions. *: p < 0.05 vs. 6 months; #: p < 0.05 vs. 12 months; n = 5. Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Age-related decline of hemoglobin-α (Hb-α) with parallel increase in brain hypoxia in the intact female mouse brain. ( A ) Representative photomicrographs from female mouse hippocampal CA1, CA3, and cerebral cortex regions showing age-related decline in Hb-α staining and parallel increase in brain hypoxia as measured by pimonidazole (hypoxyprobe-1) staining. ( B ) Semi-quantitative intensity analysis of Hb-α and pimonidazole staining in hippocampal CA1 and CA3 regions and cerebral cortex of all female mice at different ages. ( C ) Correlation coefficient analysis between Hb-α and pimonidazole in hippocampal CA1, CA3 and cortex regions. *: p < 0.05 vs. 6 months; #: p < 0.05 vs. 12 months; n = 5. Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Gender differences in neuronal hemoglobin-α (Hb-α) levels and brain hypoxia in the mouse brain in aging. Semi-quantitative intensity analysis of Hb-α ( A – C ) and pimonidazole staining ( D – F ) in hippocampal CA1 and CA3 regions and cerebral cortex of all female and male mice at different ages. *: p < 0.05 vs. female; n = 5.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Effect of hemoglobin-α knockdown on HIF-1α expression in the male mouse hippocampal CA1 and CA3 regions following global cerebral ischemia (GCI). ( A )/( a ) and ( B )/( a ) Representative photomicrographs of HIF-1α immunostaining in 3-month-old male mouse hippocampal CA1 and CA3 regions from control (empty, upper row), scramble (lenti-CRISPRi-scramble, middle row) or Hb-α knockdown (lenti-CRISPRi-HBA1/2, lower row) groups. ( A )/( b ) and ( B )/( b ) Quantification of mean immunofluorescence (IF) demonstrates a mild increase in HIF-1α expression in sham after Hb-α knockdown (*: p < 0.05, n = 6). At 7 days after GCI reperfusion (R7d), Hb-α knockdown robustly increased HIF-1α expression in both hippocampal CA1 ( A ) and CA3 regions ( B ). (*: p < 0.05, **: p < 0.01, n = 6). Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Knockdown, Expressing, Immunostaining, Control, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Effect of hemoglobin-α knockdown on expression of the HIF-1α-regulated pro-apoptotic gene, PUMA in the male mouse hippocampal CA1 and CA3 regions following global cerebral ischemia (GCI). ( A , C ) Representative photomicrographs of PUMA immunostaining in 3-month-old male mouse hippocampal CA1 ( A ) and CA3 ( C ) regions from control (empty, upper row), scramble (lenti-CRISPRi-scramble, middle row) or Hb-α knockdown (lenti-CRISPRi-HBA1/2, lower row) groups. Staining in red represents NeuN, a neuronal marker, and staining in green represents PUMA. ( B , D ) Quantification of mean immunofluorescence (IF) demonstrates a mild increase in PUMA intensity in sham after Hb-α knockdown. At 7 days after GCI reperfusion (R7d), Hb-α knockdown robustly increased PUMA intensity in the hippocampal CA1 and CA3 regions (**: p < 0.01, n = 6). Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Knockdown, Expressing, Immunostaining, Control, Staining, Marker, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Effect of hemoglobin-α knockdown on expression of the HIF-1α-regulated pro-apoptotic gene, NOXA in the male mouse hippocampal CA1 and CA3 regions following global cerebral ischemia (GCI). ( A , C ) Representative photomicrographs of NOXA immunostaining in 3-month-old male mouse hippocampal CA1 and CA3 regions from control (empty, upper row), scramble (lenti-CRISPRi-scramble, middle row) or Hb-α knockdown (lenti-CRISPRi-HBA1/2, lower row) groups. ( B , D ). Quantification of mean immuno-fluorescence (IF) demonstrates a mild increase in NOXA expression in sham after Hb-α knockdown (**: p < 0.01, n = 6). At 7 days after GCI reperfusion (R7d), Hb-α knockdown robustly increased NOXA expression in both CA1 ( A ) and CA3 regions ( C ). (**: p < 0.01, n = 6). Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Knockdown, Expressing, Immunostaining, Control, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Effect of hemoglobin-α knockdown on neurodegeneration in the male mouse hippocampal CA1 region following global cerebral ischemia (GCI). ( A ) Representative photomicrographs from male mouse hippocampal CA1 region showing staining with the neuronal marker NeuN or with the neurodegeneration marker Fluoro Jade C (FJC). ( B ) NeuN-positive cell number in hippocampal CA1 region of various groups. ( C ) Intensity analysis of FJC staining in hippocampal CA1 region of various groups. **: p < 0.01. Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Knockdown, Staining, Marker

Journal: International Journal of Molecular Sciences

Article Title: Regulation and Role of Neuron-Derived Hemoglobin in the Mouse Hippocampus

doi: 10.3390/ijms23105360

Figure Lengend Snippet: Effect of hemoglobin-α knockdown on neurodegeneration in the male mouse hippocampal CA3 region following global cerebral ischemia (GCI). ( A ) Representative photomicrographs from male mouse hippocampal CA3 region showing staining with the neuronal marker NeuN or with the neurodegeneration marker Fluoro Jade C (FJC). ( B ) NeuN-positive cell number in hippocampal CA3 region of various groups. ( C ) Intensity analysis of FJC staining in hippocampal CA3 region of various groups. *: p < 0.05, **: p < 0.01. Scale bar: 20 μm.

Article Snippet: Primary antibodies used included Hemoglobin-α (rabbit, 1:5000, Proteintech, Rosemont, IL, USA, Cat# 14537-1-AP), and GAPDH (mouse, 1:2000, Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-32233).

Techniques: Knockdown, Staining, Marker