Journal: Science Advances

Article Title: Inflammatory cytokines disrupt astrocyte exosomal HepaCAM-mediated protection against neuronal excitotoxicity in the SOD1G93A ALS model

doi: 10.1126/sciadv.adq3350

Figure Lengend Snippet: ( A ) Representative immunoblots of hSOD1G93A and misfolded SOD1 in eluted SEC fractions (pooled as indicated) of SOD1G93A ACM (10 ml). Thirty microliters of pooled fractions per lane were loaded. ( B ) Representative immunoblots of hSOD1G93A and misfolded SOD1 on SOD1G93A A-Exo. (2 μg per lane) isolated from ACM of untreated and ITC cytokine–treated SOD1G93A astrocytes. ( C ) Representative immunoblots of hSOD1G93A and misfolded SOD1 in eluted SEC fractions (pooled as indicated) of SOD1G93A NCM. Thirty microliters of pooled fractions per lane were loaded. Red dashed box highlights exosome fractions as determined by the exosome marker CD81. Representative confocal microscopy images of hCD63-GFP + and misfolded SOD1 detected by either SEDI ( D ) or A5C3 ( E ) antibody from spinal cords of AAV5-mCherry- Gfap -Cre–injected hCD63-GFP f/+ SOD1G93A + mice. [(ii) and (iii)] Two magnified fields from (i); scale bars, 20 μm. Sections used were ~1500 μm from the injection site with no mCherry signals. ( F ) Percentage of colocalization of hCD63-GFP + puncta signal with misfolded SOD1 detected by either A5C3 or SEDI antibody from spinal cords of AAV5-mCherry- Gfap -Cre–injected hCD63-GFP f/+ SOD1G93A + mice. n = 12 to 21 sections from 4 mice per group. Representative confocal microscopy images of hCD63-GFP + and misfolded SOD1 detected by either SEDI ( G ) or A5C3 ( H ) antibodies from spinal cords of AAV8-mCherry- CaMKIIa -Cre–injected hCD63-GFP f/+ SOD1G93A + mice. [(ii) and (iii)] Two magnified fields from (i); scale bars, 20 μm. Sections used were ~1000 μm from the injection site with no mCherry signals. ( I ) Percentage of colocalization of hCD63-GFP + puncta signal with misfolded SOD1 detected by either A5C3 or SEDI antibody from spinal cords of AAV8-mCherry- CaMKIIa -Cre–injected hCD63-GFP f/+ SOD1G93A + mice. n = 17 sections per three mice.

Article Snippet: The cells were blocked in 3% BSA for 30 min and incubated with the following primary antibodies overnight at 4°C: anti-ChAT (1:200, Millipore, AB144P), anti–βIII-TUBULIN (1:1000, MAB1195, R&D system), anti-RAB7 (1:100, catalog no. D9FF2, Cell Signaling Technology), anti-mouse TAU (1:500, GeneTex), anti-mouse anti-HB9 (1:50, DSHB 81.5C10), anti-Map2 (1:1000, Sigma-Aldrich, M9942), rat anti-GFAP (1:5000, zymed, 273756), and rabbit anti-GFAP (1:1000, Dako).

Techniques: Western Blot, Isolation, Marker, Confocal Microscopy, Injection

Journal: Science Advances

Article Title: Inflammatory cytokines disrupt astrocyte exosomal HepaCAM-mediated protection against neuronal excitotoxicity in the SOD1G93A ALS model

doi: 10.1126/sciadv.adq3350

Figure Lengend Snippet: ( A ) Cortical neuronal death rate following treatment with l -Glu. (100 or 1000 μM) and l -Glu. + NTg A-Exo. (1 μg). n = 23 to 31 fields (20×) per three biological replicates per group; Control, untreated cultures. Neuronal death rate was calculated by TUNEL + Hoechst + neurons divided by Hoechst + neurons. Representative images ( B ) of βIII-TUBULIN staining and quantification ( C ) of neurite beading in cortical neurons following the same treatment. White arrows in (ii), (iii), and (vi) indicate neurite beading; scale bar, 50 μm. Neurite beading index was calculated by the total number of “beads” per field (20×) divided by the βIII-TUBULIN + area in the same field. n = 23 to 31 fields (20×) per three biological replicates per group. ( D ) Schematic diagram of treatment of neuronal cultures with l -Glu. and A-Exo. (1 μg) collected from different genotype and ITC treatment combination. ( E ) Cortical neuronal death rate treated with l -Glu. (100 μM), l -Glu. + NTg or SOD1G93A A-Exo., l -Glu. + ITC-NTg, or ITC-SOD1G93A A-Exo. n = 11 to 26 fields (20×) per two biological replicates per group. All legends are shown (and the same as) in (F) due to limited space. ( F ) Quantification of neurite beading in cortical neurons following the same treatment as indicated in (E). n = 27 to 33 fields (20×) per three biological replicates per group. ( G ) Quantification of neurite beading in human iPSC–derived MNs treated with l -Glu. (200 μM) and combinations of l -Glu. with different A-Exo. (2 μg). n = 26 to 48 fields (20×) per three biological replicates per group. Data from different fields of the same biological replicate were averaged and presented in each panel. Error bars denote SEM, and P values in (E) to (G) were calculated using one-way ANOVA followed by a Tukey post hoc test. P values in (A) and (C) were calculated using two-way ANOVA followed by a Tukey post hoc test. A.U., arbitrary units.

Article Snippet: The cells were blocked in 3% BSA for 30 min and incubated with the following primary antibodies overnight at 4°C: anti-ChAT (1:200, Millipore, AB144P), anti–βIII-TUBULIN (1:1000, MAB1195, R&D system), anti-RAB7 (1:100, catalog no. D9FF2, Cell Signaling Technology), anti-mouse TAU (1:500, GeneTex), anti-mouse anti-HB9 (1:50, DSHB 81.5C10), anti-Map2 (1:1000, Sigma-Aldrich, M9942), rat anti-GFAP (1:5000, zymed, 273756), and rabbit anti-GFAP (1:1000, Dako).

Techniques: Control, TUNEL Assay, Staining, Derivative Assay

Journal: Science Advances

Article Title: Inflammatory cytokines disrupt astrocyte exosomal HepaCAM-mediated protection against neuronal excitotoxicity in the SOD1G93A ALS model

doi: 10.1126/sciadv.adq3350

Figure Lengend Snippet: Cortical neuronal death rate ( A ) and neurite beading index ( B ) in primary cortical neurons treated with l -Glu (100 μM), NTg A-Exo. + l -Glu, HepaCAM KO A-Exo. + l -Glu, HepaCAM KO + HepaCAM overexpression (OE) A-Exo. + l -Glu. Control, 1× PBS; n = 24 to 31 fields (20×) per three biological replicates per group for neuronal death rate. n = 24 to 38 fields (20×) per three biological replicates per group for neurite beading analysis. ( C ) Representative images of βIII-TUBULIN and TUNEL staining in primary cortical neurons grown on PDL + BSA or PDL + HepaCAM ECD and treated with l -Glu. (100 μM). White arrows, neurites with beading; yellow arrows, preserved axons with minimal beading. (ii) A magnified view from the box in (i); (v) a magnified view from the box in (iv); circles indicate TUNEL + neurons in (iii). Scale bar, 30 μm [(i), (iii), (iv), and (vi)]; 20 μm [(ii) and (v)]. Cortical neuronal death rate ( D ) and neurite beading index ( E ) in primary cortical neurons grown on PDL + BSA or PDL + HepaCAM ECD and treated with l -Glu. (100 μM). n = 29 to 32 fields (20×) per three biological replicates per group for neuronal death rate. n = 18 to 26 fields (20×) per two biological replicates per group for neurite beading analysis. Data from different fields of the same biological replicate were averaged and presented in each panel. Error bars denote SEM, and P values in (A), (B), (D), and (E) were calculated using one-way ANOVA followed by a Tukey post hoc test.

Article Snippet: The cells were blocked in 3% BSA for 30 min and incubated with the following primary antibodies overnight at 4°C: anti-ChAT (1:200, Millipore, AB144P), anti–βIII-TUBULIN (1:1000, MAB1195, R&D system), anti-RAB7 (1:100, catalog no. D9FF2, Cell Signaling Technology), anti-mouse TAU (1:500, GeneTex), anti-mouse anti-HB9 (1:50, DSHB 81.5C10), anti-Map2 (1:1000, Sigma-Aldrich, M9942), rat anti-GFAP (1:5000, zymed, 273756), and rabbit anti-GFAP (1:1000, Dako).

Techniques: Over Expression, Control, TUNEL Assay, Staining

Journal: Science Advances

Article Title: Inflammatory cytokines disrupt astrocyte exosomal HepaCAM-mediated protection against neuronal excitotoxicity in the SOD1G93A ALS model

doi: 10.1126/sciadv.adq3350

Figure Lengend Snippet: ( A ) Schematic representation of the microfluidic chamber system and representative images of the soma and axonal compartments. (i) Differential interference contrast (DIC) image of neurons at the soma side; (ii) DIC image of axons at the axon side; (iii) tdT + -labeled neurons by adding AAV8-CAG-tdT onto the soma side of the chamber at 10 DIV; (iv) tdT + -labeled axons at the axon side of the chamber; white lines and gray arrows indicate the ending of the soma side (i) and the beginning of the axon side (ii) of the chamber. Scale bar, 100 μm. Representative images ( B ) of tdT + axon degeneration and quantification ( C ) of axonal tdT + intensity over time following addition of l -Glu. into either soma or axon sides. White arrows, degenerating axons indicated by beading following soma side of l -Glu. treatment; yellow arrows, healthy axons even after l -Glu. treatment at the axon side; scale bar, 50 μm; n = 19 to 30 fields (10×) per three chambers per two biological replicates per group. Diagram of the experiment and representative tdT + axon images ( D ) and tdT + intensity quantification ( E ) at the axon side of the microfluidic chambers. l -Glu was added onto the soma side and all A-Exo. (NTg and HepaCAM A-Exo.) were added onto the axon side. White lines and gray arrows indicate the beginning of the axon side of the chamber. HepaCAM ECD was coated on microfluidic chambers. Time-lapse tdT + images were taken at 0-, 6-, and 22-hours after l -Glu. treatment, and the tdT fluorescent intensity was quantified. n = 19 to 39 fields (10×) per four chambers per two biological replicates per group; white arrows, degenerating axons; yellow arrows, healthy axons; scale bar, 50 μm. Error bars denote SEM. P values were calculated using two-way ANOVA followed by a Tukey post hoc test.

Article Snippet: The cells were blocked in 3% BSA for 30 min and incubated with the following primary antibodies overnight at 4°C: anti-ChAT (1:200, Millipore, AB144P), anti–βIII-TUBULIN (1:1000, MAB1195, R&D system), anti-RAB7 (1:100, catalog no. D9FF2, Cell Signaling Technology), anti-mouse TAU (1:500, GeneTex), anti-mouse anti-HB9 (1:50, DSHB 81.5C10), anti-Map2 (1:1000, Sigma-Aldrich, M9942), rat anti-GFAP (1:5000, zymed, 273756), and rabbit anti-GFAP (1:1000, Dako).

Techniques: Labeling

Journal: Investigative Ophthalmology & Visual Science

Article Title: Blue Light Damages Retinal Ganglion Cells Via Endoplasmic Reticulum Stress and Autophagy in Chickens

doi: 10.1167/iovs.66.1.3

Figure Lengend Snippet: Antibodies Used for Immunofluorescence and Western Blot

Article Snippet: Neuron-specific beta-III Tubulin (TUJ-1) , Mouse , Beyotime , AG0147 , 1:100 (IF).

Techniques: Immunofluorescence, Western Blot, Ubiquitin Proteomics

Journal: Investigative Ophthalmology & Visual Science

Article Title: Blue Light Damages Retinal Ganglion Cells Via Endoplasmic Reticulum Stress and Autophagy in Chickens

doi: 10.1167/iovs.66.1.3

Figure Lengend Snippet: BL exposure induced ER stress and impaired autophagy in the chicken retina. ( A – E ) WB bands and statistical analysis of ER stress-related protein levels in the monochromatic light treatment groups ( n = 5). ( F ) WB bands and statistical analysis of ubiquitinated proteins ( n = 5). ( G – I ) WB bands and statistical analysis of autophagy-related proteins ( n = 5). ( J ) A comparison of TRB3 expression levels in each group ( n = 5). ( K – M ) WB bands and statistical analysis of NFR2-related antioxidant pathways ( n = 5). ( N ) Schematic diagram illustrating the relationships among the unfolded protein response, autophagy, and oxidative stress. The PERK-ATF4-CHOP pathway regulates Atg5 transcription and influences autophagosome formation during the unfolded protein response. Furthermore, prolonged ER stress also activates Trb3 transcription to inhibit the degradation of P62 in autophagosomes. ( O ) Colocalization of TUJ-1 (a RGC marker) and p -PERK in the GCL and IPL under monochromatic light treatment. ( P ) Colocalization of P62 and LC3B in the chicken GCL and IPL under monochromatic light treatment. Data were presented as means ± SEM. The results were analyzed using one-way ANOVA with Sidak's multiple comparisons test; * P < 0.05 and ** P < 0.01. eIF2α, eukaryotic initiation factor 2 alpha; Ub, Ubiquitin; P62, also known as SQSTM1 (sequestosome 1); HO-1, heme oxygenase 1.

Article Snippet: Neuron-specific beta-III Tubulin (TUJ-1) , Mouse , Beyotime , AG0147 , 1:100 (IF).

Techniques: Comparison, Expressing, Marker, Ubiquitin Proteomics

Journal: Investigative Ophthalmology & Visual Science

Article Title: Blue Light Damages Retinal Ganglion Cells Via Endoplasmic Reticulum Stress and Autophagy in Chickens

doi: 10.1167/iovs.66.1.3

Figure Lengend Snippet: Both OPN4 and melatonin influence ER stress in chicken RGCs under BL in vitro. ( A ) Expression levels of genes associated with mitochondrial dynamics and damage in the retina following five weeks of monochromatic light treatment ( n = 5–9). ( B ) Fluorescent labeling of DNA fragmentation in chicken retinas. ( C ) Effects of light wavelength on the transcription levels of Opn4 orthologs in the chicken retina ( n = 5). ( D ) Protein levels of OPN4m in chicken retinas after five weeks of monochromatic light treatment ( n = 5). ( E ) BL exposure resulted in greater muscular pupil constriction in chickens ( n = 6). ( F , G ) BL exposure does not influence the kinetics of pupil contraction in chickens (one-phase association, n = 5). ( H ) Expression levels of melatonin-related proteins ( n = 5). ( I ) Localization of Mel1a in the chicken retina. ( J ) Localization of AANAT in the nuclei of RGCs ( n = 5). ( K ) Effects of the OPN4 inhibitor AA92593 on the viability of embryonic chicken retinal cells ( n = 4). The black arrows indicate the drug concentrations used in vitro. ( L ) Effects of melatonin on the viability of embryonic chicken retinal cells ( n = 4). The black arrows indicate the drug concentrations used in vitro. ( M , N ) Effects of the in vitro addition of AA92593 and melatonin on the intensity of p -PERK immune-positive signals in TUJ-1 + embryonic chicken retinal cells under BL ( n = 5). Data were presented as means ± SEM. The results were analyzed using the Kruskal-Wallis test with Dunn's multiple comparisons ( A ) or one-way ANOVA with Sidak's multiple comparisons tests ( C , D , E , H , and J ). Comparisons between drugs and their solvents were conducted using an unpaired two-tailed Student's t -test or Mann-Whitney U test ( K , L , and N ); * P < 0.05, ** P < 0.01, and **** P < 0.0001. Dnm1, dynamin 1; Opa1, optic atrophy 1; Mfn1, mitofusin-1; Mfn2, mitofusin-2; cgas, cyclic guanosine monophosphate (GMP)-AMP synthase; Sting, stimulator of interferon genes; Mel1b, melatonin receptor 2; NQO2, quinone dehydrogenase 2; Mel, melatonin.

Article Snippet: Neuron-specific beta-III Tubulin (TUJ-1) , Mouse , Beyotime , AG0147 , 1:100 (IF).

Techniques: In Vitro, Expressing, Labeling, Two Tailed Test, MANN-WHITNEY

Journal: Human Cell

Article Title: Generation of human induced pluripotent stem cell lines derived from patients of cystic biliary atresia

doi: 10.1007/s13577-024-01147-x

Figure Lengend Snippet: Pluripotency of hiPSC lines derived from cystic BA patients. A Immunocytochemistry of TUJ1, SMA, and AFP on embryoid bodies (EBs) differentiated from BA-specific hiPSCs. Scale bars, 200 µm. B Immunocytochemistry of TUJ1, SMA, and AFP on undifferentiated cBA-hiPSCs. Scale bars, 200 µm. C Sections with hematoxylin and eosin (HE) staining on teratomas derived from cBA-hiPSCs. Neural rosettes, cartilages, or intestine-like secretion tissues are indicated as derivatives of ectoderm, mesoderm, or endoderm, respectively. Red rectangles are magnified regions. Scale bars, 200 µm

Article Snippet: Differentiation marker (Ectoderm) , Mouse anti-TUJ1 , 1:250 , R and D Systems, Cat# MAB1195 , RRID:AB_357520.

Techniques: Derivative Assay, Immunocytochemistry, Staining

Journal: Human Cell

Article Title: Generation of human induced pluripotent stem cell lines derived from patients of cystic biliary atresia

doi: 10.1007/s13577-024-01147-x

Figure Lengend Snippet: Reagent information

Article Snippet: Differentiation marker (Ectoderm) , Mouse anti-TUJ1 , 1:250 , R and D Systems, Cat# MAB1195 , RRID:AB_357520.

Techniques: Immunocytochemistry, Cytometry, Marker, Nested PCR, Plasmid Preparation

Journal: ACS Omega

Article Title: OrganoLabeler: A Quick and Accurate Annotation Tool for Organoid Images

doi: 10.1021/acsomega.4c06450

Figure Lengend Snippet: Immunochemical characterization of embryoid bodies and 30 day old brain organoids generated under three-different conditions. (A) Embryoid bodies stained to demonstrate the presence of three distinct germ layers; with α-SMA (mesodermal marker), Nestin (ectodermal marker), Sox17 (endodermal marker), and DAPI (cell nucleus). (B) Brain organoid generated under static condition stained with Sox2 and Tuj1. (C) Brain organoid generated under static condition stained with N-cadherin. (D) Brain organoid generated using an orbital shaker stained with Sox2 and Tuj1. (E) Brain organoid generated using an orbital shaker stained with N-cadherin. (F) Brain organoid generated using a microfluidic chip stained with Sox2 and Tuj1. (G) Brain organoid generated using a microfluidic chip stained with N-cadherin. (Right panels: magnification of dashed zones).

Article Snippet: Subsequently, primary antibodies (α-SMA (Cell Signaling Technology, Cat no.48938S), Nestin (Proteintech, Cat no. 19483-1-AP), Sox17 (Abcam, Cat no. Ab84990), Sox2 (Bioss, Cat no. bs-0523R), Tuj1 (R&D Systems, Cat no. MAB1195), and N-cadherin (Cell Signaling Technology, Cat no. 13116T) were added according to related samples and incubated for overnight at 4 °C.

Techniques: Generated, Staining, Marker

Journal: bioRxiv

Article Title: Lrp1 facilitates infection of neurons by Jamestown Canyon virus

doi: 10.1101/2024.11.06.622176

Figure Lengend Snippet: Primary rat neurons were infected with JCV at an MOI of 0.1, 0.01, or 0.001. (A) Viral RNA was quantified at 24, 36, 48, and 60 hpi timepoints. (B) Infected or mock infected coverslips were fixed in 4% PFA and stained for JCV-N (pink) and βIII-Tubulin (green) and counterstained with Hoescht (blue). Slides were imaged at 20X using a Nikon A-1 confocal microscope. Scale bar = 250µm. (C) Western blot of uninfected primary rat neurons across different days in vitro (DIV). Blots were probed for the 85 kDa beta chain of Lrp1, βIII-Tubulin, and β-Actin. (D) Immunofluorescent microscopy of neurons 4 DIV. Coverslips were fixed with 4% PFA and stained for Lrp1 (green) and counterstained with Hoescht (blue). Slides were imaged at 10X using a Leica DMI8 inverted microscope. Scale bar = 250µm.

Article Snippet: The following primary antibodies were used in this study: mouse anti-GAPDH (1:1000; Invitrogen, MA1-16757), rabbit anti-Lrp1 (1:500; Cell Signaling, 64099S), custom rabbit anti-JCV N (1:500; Genscript, Y743THG190-16), mouse anti-βIII-tubulin (1:500; R&D Systems, MAB1195), anti-RVFV Gn Clone 4D4 (1:500; BEI Resources NR-43190) and mouse anti-β-actin (1:500; Santa Cruz Biotechnology, sc-47778).

Techniques: Infection, Staining, Microscopy, Western Blot, In Vitro, Inverted Microscopy

Journal: bioRxiv

Article Title: Lrp1 facilitates infection of neurons by Jamestown Canyon virus

doi: 10.1101/2024.11.06.622176

Figure Lengend Snippet: (A) Infectious titers at 36, 48, 60 hpi. (B) Additional images of 36 hpi, 60 hpi, and primary delete of JCV infected primary rat neurons. Coverslips were stained for JCV-N (pink) and βIII-Tubulin (green) and counterstained with DAPI (blue). Slides were imaged at 20X using a Nikon A-1 confocal microscope. Scale bar = 250µm. (C) Additional images showing Lrp1 expression in primary rat neurons across days 5, 6, and 7 in vitro. Coverslips were stained for Lrp1 (green) and counterstained with DAPI (blue). Slides were imaged at 10X using a Leica DMI8 inverted microscope. Scale bar = 250µm.

Article Snippet: The following primary antibodies were used in this study: mouse anti-GAPDH (1:1000; Invitrogen, MA1-16757), rabbit anti-Lrp1 (1:500; Cell Signaling, 64099S), custom rabbit anti-JCV N (1:500; Genscript, Y743THG190-16), mouse anti-βIII-tubulin (1:500; R&D Systems, MAB1195), anti-RVFV Gn Clone 4D4 (1:500; BEI Resources NR-43190) and mouse anti-β-actin (1:500; Santa Cruz Biotechnology, sc-47778).

Techniques: Infection, Staining, Microscopy, Expressing, In Vitro, Inverted Microscopy

Journal: bioRxiv

Article Title: Lrp1 facilitates infection of neurons by Jamestown Canyon virus

doi: 10.1101/2024.11.06.622176

Figure Lengend Snippet: Primary rat neurons were pre-treated with different concentrations of mRAP D3 for 45 minutes followed by infection with JCV at an MOI of 0.1. At 24hpi, supernatant was collected for quantification of (A) viral RNA and (B) infectious virus. (C) Coverslips were fixed with 4% PFA and stained for JCV-N (pink) and βIII-Tubulin (green) and counterstained with Hoescht (blue). Slides were imaged at 10X using a Leica DMI8 inverted microscope. Scale bar = 250µm. Statistics determined by one-way ANOVA. **** p<0.0001.

Article Snippet: The following primary antibodies were used in this study: mouse anti-GAPDH (1:1000; Invitrogen, MA1-16757), rabbit anti-Lrp1 (1:500; Cell Signaling, 64099S), custom rabbit anti-JCV N (1:500; Genscript, Y743THG190-16), mouse anti-βIII-tubulin (1:500; R&D Systems, MAB1195), anti-RVFV Gn Clone 4D4 (1:500; BEI Resources NR-43190) and mouse anti-β-actin (1:500; Santa Cruz Biotechnology, sc-47778).

Techniques: Infection, Virus, Staining, Inverted Microscopy

Journal: bioRxiv

Article Title: Lrp1 facilitates infection of neurons by Jamestown Canyon virus

doi: 10.1101/2024.11.06.622176

Figure Lengend Snippet: (A) vRNA from K256A/K270E mutant mRAP D3 treated cells. (B) Additional images of mRAP D3 treatment of primary rat neurons, including primary delete. Coverslips were stained for JCV-N (pink) and βIII-Tubulin (green) and counterstained with Hoescht (blue). Slides were imaged at 10X using a Leica DMI8 inverted microscope. Scale bar = 250µm. Statistics determined by one-way ANOVA. *p=0.0315, ***p=0.0001, ****p<0.0001.

Article Snippet: The following primary antibodies were used in this study: mouse anti-GAPDH (1:1000; Invitrogen, MA1-16757), rabbit anti-Lrp1 (1:500; Cell Signaling, 64099S), custom rabbit anti-JCV N (1:500; Genscript, Y743THG190-16), mouse anti-βIII-tubulin (1:500; R&D Systems, MAB1195), anti-RVFV Gn Clone 4D4 (1:500; BEI Resources NR-43190) and mouse anti-β-actin (1:500; Santa Cruz Biotechnology, sc-47778).

Techniques: Mutagenesis, Staining, Inverted Microscopy

Journal: bioRxiv

Article Title: Lrp1 facilitates infection of neurons by Jamestown Canyon virus

doi: 10.1101/2024.11.06.622176

Figure Lengend Snippet: Primary rat neurons were pre-treated with different concentrations of recombinant, soluble RVFV Gn for 45 minutes followed by infection with JCV at an MOI of 0.1. At 24hpi, supernatant was collected for quantification of (A) viral RNA. Cells were lysed in RIPA buffer and to assess protein levels via Western blot (B). Western blots were probed for RVFV Gn, JCV-N, and β-actin. (C) Coverslips were fixed with 4% PFA and stained for JCV-N (pink) and βIII-Tubulin (green) and counterstained with DAPI (blue). Slides were imaged at 10X using a Leica DMI8 inverted microscope. Scale bar = 250µm. Statistics determined by one-way ANOVA. **p=0.0016, ***p=0.0008.

Article Snippet: The following primary antibodies were used in this study: mouse anti-GAPDH (1:1000; Invitrogen, MA1-16757), rabbit anti-Lrp1 (1:500; Cell Signaling, 64099S), custom rabbit anti-JCV N (1:500; Genscript, Y743THG190-16), mouse anti-βIII-tubulin (1:500; R&D Systems, MAB1195), anti-RVFV Gn Clone 4D4 (1:500; BEI Resources NR-43190) and mouse anti-β-actin (1:500; Santa Cruz Biotechnology, sc-47778).

Techniques: Recombinant, Infection, Western Blot, Staining, Inverted Microscopy

Journal: bioRxiv

Article Title: Lrp1 facilitates infection of neurons by Jamestown Canyon virus

doi: 10.1101/2024.11.06.622176

Figure Lengend Snippet: (A) Additional images of RVFV Gn treatment of primary rat neurons, including primary delete. Coverslips were stained for JCV-N (pink) and βIII-Tubulin (green) and counterstained with Hoescht (blue). Slides were imaged at 10X using a Leica DMI8 inverted microscope. Scale bar = 250µm.

Article Snippet: The following primary antibodies were used in this study: mouse anti-GAPDH (1:1000; Invitrogen, MA1-16757), rabbit anti-Lrp1 (1:500; Cell Signaling, 64099S), custom rabbit anti-JCV N (1:500; Genscript, Y743THG190-16), mouse anti-βIII-tubulin (1:500; R&D Systems, MAB1195), anti-RVFV Gn Clone 4D4 (1:500; BEI Resources NR-43190) and mouse anti-β-actin (1:500; Santa Cruz Biotechnology, sc-47778).

Techniques: Staining, Inverted Microscopy