Journal: MethodsX

Article Title: Feline leukocyte immunophenotyping: an optimised whole-blood flow cytometry protocol

doi: 10.1016/j.mex.2026.103869

Figure Lengend Snippet: Representative leukocyte-gated histograms illustrating antibody titration for feline leukocyte immunophenotyping. All histograms display singlet leukocytes, defined by FSC-A versus SSC-A morphological gating, followed by FSC—H versus FSC-A singlet discrimination. Titration was performed for CD18, CD21, CD45R, CD4, CD5 and CD8 monoclonal antibodies using three antibody volumes: 10 µL (1:10 dilution), 5.0 µL (1:20 dilution), 3.0 µL (1:33 dilution) and 1.5 µL (1:66 dilution). Minimal working volumes were selected based on optimal signal-to-noise ratios.

Article Snippet: Step 2 – Leukocytes extracellular staining Materials • EDTA Whole blood sample ± Transfix® • 200 μl pipettes • 100 μl pipettes • 10 μl pipettes • Flow cytometry tubes • Permanent marker • Cytometer tube rack Reagents • Monoclonal antibodies: ○ CD5 Anti-cat – clone FE1.1B11 (BIO-RAD®) ○ CD4 Anti-cat – clone vpg34 (BIO-RAD®) ○ CD8 Anti-cat alpha/beta purified – clone vpg9 (BIO-RAD®) ○ Rat Anti-Mouse IgG1 – clone X56 (BIO-RAD®) ○ CD18 Mouse Anti-Dog – clone CA1.4E9 (BIO-RAD®) ○ CD21 Mouse Anti-Dog – clone CA2.1D6 (BIO-RAD®) ○ CD45R Rat Anti-Mouse – clone RA3–6B2 (BIO-RAD®) • 10x Red blood cells (RBC) lysis buffer solution (BD FACSTM lysing solution) • PBS 1% solution Equipment • Countess TM 3 (Thermo Fisher Scientific, USA) • Freezer • Dark incubation chamber • Timer • Vortex (MX-S®, China) • Centrifuge (model 5810R, Eppendorf®, Germany) • Flow cytometry analyser BD FACSCanto II (Becton Dickinson (BD), San Jose, USA) Methods 1.

Techniques: Titration, Bioprocessing

Journal: Bioactive Materials

Article Title: Apolipoprotein E knockout attenuates vascular graft fibrosis by reducing profibrotic macrophage formation through low-density lipoprotein receptor related protein 1

doi: 10.1016/j.bioactmat.2026.01.029

Figure Lengend Snippet: Involvement of profibrotic macrophages in vascular regeneration after graft implantation in vivo . (a) UMAP of macrophages in native aortas and regenerated aortas across different time points after graft implantation in vivo . (b) Dot plots of profibrotic macrophage marker genes (Ctsd, Spp1, Gpnmb, Lgals3, and Fabp5) expressed in different subgroups of macrophages. (c) Percentage of cluster 2 (C2) macrophages in native aortas and regenerated aortas across different time points after graft implantation in vivo . (d) UMAP of expression of Ctsd, Spp1, Gpnmb, Lgals3, and Fabp5 in macrophages in native aortas and regenerated aortas across different time points after graft implantation in vivo . (e) Immunofluorescence staining of CD68 and CTSD in regenerated aortas across different time points after graft implantation in vivo . L indicates lumens. Arrow heads indicate double positively stained cells. (f) WB results of levels of CTSD and SPP1 in native and regenerated aortas across different time points after graft implantation in vivo and quantification of the levels of CTSD and SPP1. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each time point and each group, five different samples from five different animals were analyzed (n = 5).

Article Snippet: The following primary antibodies were used in this study: APOE (Invitrogen, PA5-78803, 1:200 dilution), COL I (abcam, ab270993, 1:200 dilution), COL III (abcam, ab6310, 1:200 dilution), LUM (abcam, ab252925, 1:200 dilution), elastin (abcam, ab307150, ab307150, 1:200 dilution), eNOS (abcam, ab5589, 1:200 dilution), αSMA (abcam, ab7817, 1:200 dilution), Fibronectin (FN, abcam, ab268020, 1:200 dilution), CTSD (CST, 74089S, 1:200 dilution), CD68 (BioRad, MCA341GA, 1:100 dilution), LRP1 (Invitrogen, PA5-101013, 1:200 dilution), Ki67 (Servicebio, GB111141 , 1:200 dilution), and IGF1 (Invitrogen, MA5-18035, 1:200 dilution).

Techniques: In Vivo, Marker, Expressing, Immunofluorescence, Staining

Journal: Bioactive Materials

Article Title: Apolipoprotein E knockout attenuates vascular graft fibrosis by reducing profibrotic macrophage formation through low-density lipoprotein receptor related protein 1

doi: 10.1016/j.bioactmat.2026.01.029

Figure Lengend Snippet: APOE KO reducing profibrotic macrophage formation during vascular regeneration. (a) UMAP of macrophages in native aortas from WT and Apoe −/− rats, heatmap of C2 scores in the UMAP of macrophages in the native aortas, and percentage of C2 cells in macrophages in the native aortas. UMAP of macrophages in regenerated aortas after graft implantation in WT and Apoe −/− rats, heatmap of C2 scores in the UMAP of macrophages in the regenerated aortas, and percentage of C2 cells in macrophages in the regenerated aortas on Day 30 (b) and Day 90 (c). (d) Immunofluorescence staining of CD68 and CTSD in regenerated aortas 30 and 90 days after graft implantation in WT and Apoe−/− rats. (e) Quantification of CD68 and CTSD double positive cells in regenerated aortas on Day 30 and Day 90. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each time point and each group, five different images from five different samples were analyzed (n = 5). (f) WB results of APOE, CTSD and SPP1 levels in regenerated aortas after graft implantation in WT and Apoe −/− rats for 30 and 90 days. (g) Quantification of levels of APOE, CTSD and SPP1 in regenerated aortas on Day 30 and Day 90. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each time point and each group, five different samples from five different animals were analyzed (n = 5). (h) WB results of APOE, CTSD and SPP1 levels in WT and APOE KO macrophages after their culture on PCL scaffolds for 48 h. (i) Quantification of levels of APOE, CTSD and SPP1 in WT and APOE KO macrophages after their culture on PCL scaffolds for 48 h ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, unpaired t -test. For each time point and each group, three different samples were analyzed (n = 3). (j) Immunofluorescence staining of APOE and CD68, CTSD and CD68, SPP1 and CD68, respectively, in WT and APOE KO macrophages after their culture on PCL scaffolds for 48 h.

Article Snippet: The following primary antibodies were used in this study: APOE (Invitrogen, PA5-78803, 1:200 dilution), COL I (abcam, ab270993, 1:200 dilution), COL III (abcam, ab6310, 1:200 dilution), LUM (abcam, ab252925, 1:200 dilution), elastin (abcam, ab307150, ab307150, 1:200 dilution), eNOS (abcam, ab5589, 1:200 dilution), αSMA (abcam, ab7817, 1:200 dilution), Fibronectin (FN, abcam, ab268020, 1:200 dilution), CTSD (CST, 74089S, 1:200 dilution), CD68 (BioRad, MCA341GA, 1:100 dilution), LRP1 (Invitrogen, PA5-101013, 1:200 dilution), Ki67 (Servicebio, GB111141 , 1:200 dilution), and IGF1 (Invitrogen, MA5-18035, 1:200 dilution).

Techniques: Immunofluorescence, Staining

Journal: Bioactive Materials

Article Title: Apolipoprotein E knockout attenuates vascular graft fibrosis by reducing profibrotic macrophage formation through low-density lipoprotein receptor related protein 1

doi: 10.1016/j.bioactmat.2026.01.029

Figure Lengend Snippet: APOE/LRP1 interaction promoting profibrotic macrophage formation during vascular regeneration after graft implantation in vivo . (a) Immunoprecipitation (IP) following mass spectrometry (MS) to screen potential receptors of APOE on surfaces of macrophages. (b) Co-immunoprecipitation (Co-IP) to confirm interaction between APOE and LRP1. (c) Immunofluorescence staining of CD68 and LRP1 in regenerated aortas across different time points. (d) Immunofluorescence staining of APOE and LRP1 in WT macrophages 48 h after their culture on PCL scaffolds. (e) WB results of LRP1, APOE, CTSD and SPP1 levels in WT macrophages cultured on tissue culture plates (negative control, NC) or PCL scaffolds (PCL) for 48 h prior to treatment with shRNA ADV-shRNA(NC) or ADV-shRNA(Lrp1) for 24 h. Quantification of levels of LRP1 (f), APOE (g), CTSD (h) and SPP1 (i) in WT macrophages cultured on tissue culture plates or PCL scaffolds treated with shRNA ADV-shRNA(NC) or ADV-shRNA(Lrp1). ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, N.S. indicates non-significant. Tukey's post-hoc test. For each group, three different samples were analyzed (n = 3). (j) Flow cytometry analysis of CTSD positive cells in WT macrophages cultured on tissue culture plates (negative control, NC) or PCL scaffolds (PCL) for 48 h prior to treatment with ADV-shRNA(NC) or ADV-shRNA(Lrp1) for 24 h and quantification of percentage of CTSD positive cells in WT macrophages in each group. ∗ indicates p < 0.05, Tukey's post-hoc test. For each group, three independent experiments were repeated, and results were analyzed (n = 3).

Article Snippet: The following primary antibodies were used in this study: APOE (Invitrogen, PA5-78803, 1:200 dilution), COL I (abcam, ab270993, 1:200 dilution), COL III (abcam, ab6310, 1:200 dilution), LUM (abcam, ab252925, 1:200 dilution), elastin (abcam, ab307150, ab307150, 1:200 dilution), eNOS (abcam, ab5589, 1:200 dilution), αSMA (abcam, ab7817, 1:200 dilution), Fibronectin (FN, abcam, ab268020, 1:200 dilution), CTSD (CST, 74089S, 1:200 dilution), CD68 (BioRad, MCA341GA, 1:100 dilution), LRP1 (Invitrogen, PA5-101013, 1:200 dilution), Ki67 (Servicebio, GB111141 , 1:200 dilution), and IGF1 (Invitrogen, MA5-18035, 1:200 dilution).

Techniques: In Vivo, Immunoprecipitation, Mass Spectrometry, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Cell Culture, Negative Control, shRNA, Flow Cytometry

Journal: Bioactive Materials

Article Title: Apolipoprotein E knockout attenuates vascular graft fibrosis by reducing profibrotic macrophage formation through low-density lipoprotein receptor related protein 1

doi: 10.1016/j.bioactmat.2026.01.029

Figure Lengend Snippet: Downregulation of APOE by AAV ameliorating fibrosis during vascular regeneration after graft implantation in vivo . (a) Illustration of a strategy of adventitial delivery of AAV-shRNA(Apoe) to inhibit APOE levels in regenerated aortas after graft implantation in vivo . Two weeks after graft implantation in vivo , AAV-shRNA(Apoe) were injected into the adventitia of the regenerated aortas, which were then harvested for analysis three weeks later. (b) M mode images of ultrasound detection of regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. Arrow heads indicate movement of vascular walls. (c) Tensile tests of regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. (d) Quantification of RI, PI, and compliance of regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each group, six different images from six different animals were analyzed (n = 6). (e) Quantification of elastic modulus of regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each group, six different images from six different animals were analyzed (n = 6). (f) H&E, MTC and EVG staining of regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. (g) Immunofluorescence staining of COL I, COL III, elastin, αSMA, and eNOS in regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. L indicates lumens. Arrow heads indicate capillaries. Quantification of adventitia thickness (h), collagen positive areas according to MTC staining (i), elastin positive areas according to EVG staining (j), COL I positive areas (k), COL III positive areas (l), and number of capillaries (m) in adventitial areas of regenerated aortas. (n) Immunofluorescence staining of CTSD and CD68 in regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks. (o) CD68 and CTSD double positive cells in regenerated aortas. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each group, six different samples from six different animals were analyzed (n = 6). (p) WB results of APOE, CTSD and SPP1 levels in regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks and quantification of levels of APOE, CTSD and SPP1 in regenerated aortas. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each group, six different samples from six different animals were analyzed (n = 6). (q) Quantification of IGF-1 concentrations in regenerated aortas treated with PBS, AAV-shRNA(NC), and AAV-shRNA(Apoe) for 3 weeks by ELISA. ∗∗ indicates p < 0.01, Tukey's post-hoc test. For each group, six different samples from six different animals were analyzed (n = 3).

Article Snippet: The following primary antibodies were used in this study: APOE (Invitrogen, PA5-78803, 1:200 dilution), COL I (abcam, ab270993, 1:200 dilution), COL III (abcam, ab6310, 1:200 dilution), LUM (abcam, ab252925, 1:200 dilution), elastin (abcam, ab307150, ab307150, 1:200 dilution), eNOS (abcam, ab5589, 1:200 dilution), αSMA (abcam, ab7817, 1:200 dilution), Fibronectin (FN, abcam, ab268020, 1:200 dilution), CTSD (CST, 74089S, 1:200 dilution), CD68 (BioRad, MCA341GA, 1:100 dilution), LRP1 (Invitrogen, PA5-101013, 1:200 dilution), Ki67 (Servicebio, GB111141 , 1:200 dilution), and IGF1 (Invitrogen, MA5-18035, 1:200 dilution).

Techniques: In Vivo, shRNA, Injection, Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: The Journal of General Physiology

Article Title: Beat-locked ATP microdomains in the sinoatrial node map a Ca 2+ -timed energetic hierarchy and regional pacemaker roles

doi: 10.1085/jgp.202513874

Figure Lengend Snippet: Superior SA node myocytes exhibit elevated diastolic ATP and metabolic flux compared with the inferior region. (A) 3D segmented maximum-intensity projection of a whole-mount SA node immunolabeled for CD31 (vasculature, red) and cyto-iATP (myocytes, green). The dashed line denotes the boundary between superior and inferior regions. (B) Image-processing workflow illustrating merged maximum-intensity projections, binary segmentation masks, and extraction of grayscale cyto-iATP signals used for quantitative analysis. (C) Mean cyto-iATP fluorescence intensity per myocyte, grouped by region ( N = 5 mice per region), reporting expression levels of the EGFP-tagged cyto-iATP sensor. (D) Live confocal imaging of cyto-iATP signals showing representative line-scan images and corresponding normalized fluorescence traces (F/F 0 ) from superior and inferior regions. (E and F) Summary quantification of cyto-iATP signal mass rate (E) and estimated diastolic [ATP] i (F). P values are shown above comparisons. Large circles denote per-animal means; small circles indicate individual biological replicates. N represents the number of independent mice.

Article Snippet: For immunolabeling, SA nodes were incubated for 48 h at 4°C with a goat anti-mouse CD31 primary antibody (1:50, AF3628; R&D Systems).

Techniques: Immunolabeling, Extraction, Fluorescence, Expressing, Imaging