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

Journal: PLoS ONE

Article Title: Expression and Purification of Recombinant Hemoglobin in Escherichia coli

doi: 10.1371/journal.pone.0020176

Figure Lengend Snippet: As shown in Panel A, the pGM-HαHβ plasmid system contains a cassette of tandemly arrayed α- and β-globin genes with a Shine-Dalgarno ribosomal binding site as a spacer DNA. The MAP cassette is located downstream of β-globin. As shown in panel B, the pCO-MAP plasmid contains a MAP cassette under the control of a T7 promoter along with a kanamycin resistance gene.

Article Snippet: The α/β-globin and MAP cassettes were synthesized by Genscript (Piscataway, NJ, USA).

Techniques: Plasmid Preparation, Binding Assay, Control

Journal: PLoS ONE

Article Title: Expression and Purification of Recombinant Hemoglobin in Escherichia coli

doi: 10.1371/journal.pone.0020176

Figure Lengend Snippet: The relative fractions of methionine and valine in the N-terminal residue positions are shown for three experimental conditions: (A) BL21Star™ (DE3) at 12°C for 24 hr, (B) BL21Star™ (DE3) at 30°C for 4 hr with co-expression of pCO-MAP expression plasmid, and (C) JM109 (DE3) at 28°C for 16 hr with co-expression of pCO-MAP expression plasmid. The efficiency of the MAP enzyme in cleaving the N-terminal methione residues from the α- and β-chain subunits is indicated by the relative fraction of methionine vs. valine in the N-terminal residue position. When the percentage of globin chains with NA1 valine exceeds 95%, this indicates that the post-translational modification has been carried out properly.

Article Snippet: The α/β-globin and MAP cassettes were synthesized by Genscript (Piscataway, NJ, USA).

Techniques: Residue, Expressing, Plasmid Preparation, Modification