rabbit anti apoe antibody  (Bio-Rad)

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Serum factors create species-specific barriers to hepatic gene transfer by lipid nanoparticles in liver-humanized mice

doi: 10.1016/j.omtm.2025.101470

Figure Lengend Snippet: Comparative histopathology and putative LNP receptor expression in chimeric NSG-PiZ and FRG mouse livers Livers from liver-humanized NSG-PiZ or FRG mice at 24- or 48-h post treatment with PBS, or LNP1–3 formulated with DasherGFP mRNA (A and B). Paraffin-embedded sections from representative livers were stained with H&E (A). Scale bar, 100 μm. Frozen sections from representative livers were stained with Oil red O and counterstained with hematoxylin (B). Scale bars, 1 mm (inset image) and 100 μm (main image). Dashed lines indicate approximate borders between human and mouse hepatocytes. H, human hepatocytes; M, mouse hepatocytes. snRNA-seq and scRNA-seq analysis of putative LNP receptor mRNA levels in hepatocytes of chimeric FRG and NSG-PiZ mice (C). (D) Detection of ALB , A PO E , LDLR , ASGR1 , and ASGR2 mRNA in human and mouse hepatocytes from NSG-PiZ and FRG mice. (E) The percentage of total reads per cell for ALB , APOE , LDLR , ASGR1 , and ASGR2 in human (H) or mouse (M) hepatocytes determined by scRNA-seq (NSG-PiZ) or snRNA-seq (FRG). For the scRNA-seq analysis, cells were pooled from four low engrafted chimeric NSG-PiZ mice prior to single-cell analysis. For the snRNA-seq analysis, reads were pooled from two highly engrafted chimeric FRG mice or two low engrafted chimeric FRG mice from a previously published dataset (Bioproject: PRJNA857105; GEO: GSM6319631 , GSM6319632 , GSM6319637 , GSM6319638 ; Cabanes-Creus et al. ). FRG reads were processed as previously described (Cabanes-Creus et al. ) and scRNA-seq reads were processed as described in the methods. (C) was created using Biorender.

Article Snippet: Serum from chimeric NSG-PiZ mice (HuAlb >1 mg/mL) was depleted of ApoE via immunoprecipitation using a rabbit anti-human ApoE polyclonal antibody (cat # VPA00665 , Bio-Rad, Hercules, CA) and protein G spin trap columns (Cat #28903134, Marlborough, MA) according to the manufacturer’s instructions.

Techniques: Histopathology, Expressing, Staining, Single-cell Analysis

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Serum factors create species-specific barriers to hepatic gene transfer by lipid nanoparticles in liver-humanized mice

doi: 10.1016/j.omtm.2025.101470

Figure Lengend Snippet: NSG-PiZ serum factors impact LNP transfection of human hepatocytes (A) Flow cytometry and immunofluorescence analysis of GFP expression in mouse and human (HLA+ or hCK18+) hepatic cells from chimeric NSG-PiZ mice 24 h after intravenous delivery of SM-102 LNPs (1.3 mg/kg). Chimeric NSG-PiZ mice were administered PBS ( n = 2) or SM-102 LNPs ( n = 4). Representative scatterplots and histograms show relative GFP expression in human or mouse cells for two PBS- and three SM-102 LNP-treated mice along with mouse HuAlb concentrations prior to LNP administration. Representative immunofluorescence staining of paraffin sections for the fourth SM-102 LNP-treated mouse with antibodies specific for GFP (green) and hCK18 (red). (B) serum impacts transfection of PHH in vitro . SM-102 LNP was preincubated with the indicated serum for 15 min at room temperature (RT) prior to addition to media. GFP expression was imaged 6 h after after LNP administration. (C) Inhibition of PHH transfection by chimeric NSG-PiZ mouse serum. SM-102 LNPs, LP01 LNPs, or ALC0315 LNPs were pre-treated with normal mouse serum for 15 min at RT prior to addition to media containing 2% chimeric serum. GFP expression was imaged 6 h after after LNP administration. (D) Inhibition of PHH transfection by recombinant human ApoE. SM-102 LNP was pre-treated with normal mouse serum for 15 min at RT prior to addition to media containing recombinant human ApoE at the indicated concentration. (E) Inhibition of PHH transfection by ApoE depleted chimeric NSG-PiZ mouse serum. Western blot for ApoE using chimeric NSG-PiZ serum, anti-ApoE depleted chimeric NSG-PiZ serum, or control anti-Flag depleted chimeric NSG-PiZ serum (left). SM-102 LNP was pre-treated with normal mouse serum for 15 min at RT prior to addition to media containing 2% chimeric NSG-PiZ serum deleted of ApoE. GFP expression was imaged 6 h after after LNP administration. (F) Inhibition of PHH transfection by naive NSG-PiZ mouse serum. SM-102 LNP was pre-treated with normal mouse serum for 15 min at RT prior to addition to media containing 2% chimeric or naive NSG-PiZ serum. GFP expression was imaged 6 h after after LNP administration. (G) NSG mouse serum supports SM-102 transfection of PHH. SM-102 LNP was pre-treated with NSG serum or chimeric NSG-PiZ serum for 15 min at RT prior to addition to media. GFP expression was imaged 6 h after LNP administration. The experiments shown in (C–F) were performed concurrently and for comparison, the same representative image for SM-102 transfection of PHH after pre-incubation with normal mouse serum is shown as the upper left image within each panel.

Article Snippet: Serum from chimeric NSG-PiZ mice (HuAlb >1 mg/mL) was depleted of ApoE via immunoprecipitation using a rabbit anti-human ApoE polyclonal antibody (cat # VPA00665 , Bio-Rad, Hercules, CA) and protein G spin trap columns (Cat #28903134, Marlborough, MA) according to the manufacturer’s instructions.

Techniques: Transfection, Flow Cytometry, Immunofluorescence, Expressing, Staining, In Vitro, Inhibition, Recombinant, Concentration Assay, Western Blot, Control, Comparison, Incubation

Journal: The journal of headache and pain

Article Title: Astrocytic spermidine insufficiency contributes to enhanced pain sensitivity associated with ApoE4.

doi: 10.1186/s10194-025-02054-8

Figure Lengend Snippet: Fig. 1 ApoE4 enhanced neuropathic pain sensitivity in mice. (A) Schematic of experimental design, SNI surgery, and von Frey filament test. (B) Mechani cal thresholds of ApoE3-TR mice and ApoE4-TR mice before and after SNI. n = 10 mice per group. Cohen’s d for ApoE3-TR vs. ApoE4-TR post-SNI: 1.26 (D7), 1.46 (D14), 1.29 (D28). (C) Schematic diagram of DigiGait™ analysis. (D, E) Quantification of foot swing (D) and foot cycle (E) during DigiGait™ analysis of ApoE3-TR mice and ApoE4-TR mice before and 14 days after SNI. n = 6 mice per group. Cohen’s d = 2.71 for foot swing and 2.06 for foot cycle (ApoE3-TR SNI vs. ApoE4-TR SNI). (F) Schematic diagram of mechanical pain-evoked Ca2+ signals recording. CMOS, complementary metal oxide semiconductor. (G) Heatmap showing the GCaMP6f fluorescence changes in the superficial dorsal horn of the spinal cord in ApoE3-TR and ApoE4-TR mice following pain stimulation 14 days after SNI. n = 10 mice per group. (H) Peri-event plot of GCaMP6f ΔF/F after pain stimulation. (I) Quantification of the mean AUC value for ApoE3-TR and ApoE4-TR mice. n = 10 mice per group. (J) Representative images of the spinal dorsal horn labeled with anti-ApoE (red), anti-GFAP (green), and anti-Iba1 (gray) antibodies in ApoE3-TR and ApoE4-TR mice 14 days after SNI. Scale bar, 30 μm (left), 10 μm (right). All data are expressed as mean ± SEM. Statistical comparisons were conducted with two-way ANOVA followed by Bonferroni’s post hoc test (B); one-way ANOVA followed by Tukey’s post hoc test (D, E); and unpaired Student’s t-test (I). *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: Blocking was carried out using 5% BSA and 1% Triton X-100 in PBS for 30 min. Primary antibodies diluted in blocking buffer (1:500) were applied and incubated overnight at 4 °C at the following concentrations: ApoE (Cell Signaling Technology, Cat# 13366, RRID: AB_2798191), GFAP (Millipore, Cat# MAB360, RRID: AB_11212597), NeuN (Cell Signaling Technology, Cat# 24307, RRID: AB_2651140), and Iba1 (Abcam, Cat# ab5076, RRID: AB_2224402).

Techniques: Fluorescence, Labeling

Journal: The journal of headache and pain

Article Title: Astrocytic spermidine insufficiency contributes to enhanced pain sensitivity associated with ApoE4.

doi: 10.1186/s10194-025-02054-8

Figure Lengend Snippet: Fig. 2 Astrocytic ApoE4 in spinal dorsal horn contributed to the increased pain sensitivity. (A) Schematic of experimental design, AAV administration, and transgenic mice information. (B) Western blots showing the expression of Flag and ApoE after the injection of AAV-GFAP-EGFP-Cre virus. n = 3 indepen dent biological replicates per group. (C) Representative images of the spinal dorsal horn labeled with anti-ApoE (red) and anti-GFAP (gray) in LSL-hApoE3 and LSL-hApoE4 mice injected with AAV-GFAP-EGFP-Cre virus. Scale bar, 50 μm (left), 10 μm (right). (D) Mechanical thresholds before and after SNI in LSL-hApoE3 and LSL-hApoE4 mice following virus injection. n = 6 mice per group. Cohen’s d for LSL-hApoE3 vs. LSL-hApoE4 post-SNI: 1.74 (D7), 0.96 (D14), 1.48 (D28). (E, F) Quantification of foot swing (E) and foot cycle (F) during DigiGait™ analysis of LSL-hApoE3 mice and LSL-hApoE4 mice after viral injec tion, before and 14 days after the SNI. n = 6 mice per group. Cohen’s d = 2.26 for foot swing and 2.28 for foot cycle (LSL-hApoE4 SNI vs. LSL-hApoE4 SNI). (G) Experimental scheme of the fiber photometry analysis. (H) Heatmap of mechanical pain-evoked Ca2+ signals from LSL-hApoE3 and LSL-hApoE4 mice following injection of GFAP-Cre and GCaMP6f viruses. n = 10 mice per group. (I) Peri-event plot of GCaMP6f ΔF/F after pain stimulation. (J) Quantification of mean AUC value for LSL-hApoE3 and LSL-hApoE4 mice. n = 10 mice per group. All data are expressed as mean ± SEM. Statistical comparisons were con ducted with two-way ANOVA followed by Bonferroni’s post hoc test (D); one-way ANOVA followed by Tukey’s post hoc test (E, F); and unpaired Student’s t-test (J). *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: Blocking was carried out using 5% BSA and 1% Triton X-100 in PBS for 30 min. Primary antibodies diluted in blocking buffer (1:500) were applied and incubated overnight at 4 °C at the following concentrations: ApoE (Cell Signaling Technology, Cat# 13366, RRID: AB_2798191), GFAP (Millipore, Cat# MAB360, RRID: AB_11212597), NeuN (Cell Signaling Technology, Cat# 24307, RRID: AB_2651140), and Iba1 (Abcam, Cat# ab5076, RRID: AB_2224402).

Techniques: Transgenic Assay, Western Blot, Expressing, Injection, Virus, Labeling