macrophages  (Bio-Rad)

Journal: Advanced Science

Article Title: Spatial Transcriptomics Reveals a T Cell‐Mediated Microglial Activation Axis of Immune Checkpoint Inhibition‐Related Adverse Events in the Brain

doi: 10.1002/advs.77733

Figure Lengend Snippet: Depletion of T cells prevents ICI treatment‐induced microglial activation. (A) Experimental Design: Transgenic T‐DTR mice received diphtheria toxin A (DT) either 2 days before or 7 days after ICI treatment (anti‐CTLA‐4 and anti‐PD‐1). ICI treatments were given for 3 weeks (see Materials & Methods for details). WT (C57Bl/6) mice receiving either ITC or ICI treatment were used for comparison. Brains were collected at 72 h after completion of ICI treatments. (B) Representative dual‐immunofluorescence confocal z‐stacks depicting microglial activation (CD68, magenta, IBA1, green, and DAPI nuclear counterstaining) from T‐DTR and WT mouse hippocampus. (C) A 3D surface rendering of CD68‐IBA1 + activated microglia from the WT + ICI group (white arrows). (D) Volumetric quantification of CD68 and IBA1 immunoreactivity showing ICI treatment elevated microglial activation in WT + ICI group compared to WT + Vehicle. T cell depletion (T‐DTR mice) did not lead to microglial activation. Data are presented as Mean ± SEM, N = 6–8 mice/group. P ‐values were derived from ANOVA and Dunn's multiple comparisons test. Scale bar, 50 µm (A,B).

Article Snippet: Primary antibodies to characterize microglial activation include rat anti‐mouse CD68 (1:500, Bio‐Rad, Cat# MCA1957; RRID: AB_322219), and rabbit anti‐IBA1 (1:500, Wako, Cat# 019–19741; RRID: AB_839504).Secondary antibodies used for CD68 marker 1:1000; Goat anti Rat AF 647 (Abcam; Cat. ab150159), and 1:500; Goat anti Rabbit AF 488 (FisherSci; Cat. a11008) for IBA1 marker] For staining, tissue sections were first washed 3 times in phosphate‐buffered saline (1 × PBS, 100 m m , pH 7.4, FisherSci) and, when necessary, antigen retrieval was performed using citrate buffer incubation (10 m m , pH 6 with 0.05% Tween‐20, Sigma) at 70°C for 45 min. Tissues were then incubated in borate buffer (pH 8.5, 100 m m , Sigma, Cat. B0394) for 10 min, followed by three 5‐min washes in 1 × PBS.

Techniques: Activation Assay, Transgenic Assay, Comparison, Immunofluorescence, Confocal, Derivative Assay

Journal: EJNMMI Research

Article Title: TSPO PET selectively detects the denervated skeletal muscle district remodeling after experimental peripheral nerve injury

doi: 10.1186/s13550-026-01497-7

Figure Lengend Snippet: Ex vivo validation of inflammation and TSPO upregulation in denervated skeletal muscle . Representative hematoxylin and eosin (H&E)—stained sections ( A ) and CD68 immunofluorescence images ( B ) of tibialis anterior muscle collected one-week after surgery from contralateral hindlimb (Control), sham-operated (Sham), and homolateral hindlimb of injured sciatic nerve (Crush). CD68-positive macrophages are shown in red; nuclei are counterstained with DAPI (blue). Scale bar: 50 µm. ( C ) Quantification of CD68-positive area, expressed as percentage of total tissue area. Transiently denervated muscles showed significantly greater macrophage infiltration than control and sham groups. Each dot represents one biological replicate (one muscle per animal). Statistical analysis was performed using Kruskal–Wallis test, followed by Dunn’s post hoc test (*p < 0.05; **p < 0.01)

Article Snippet: Sections were then incubated overnight at 4 °C with primary antibodies against laminin α-2 (Santa Cruz Biotechnology, sc-59854, 1:200), TSPO (Thermo Fisher Scientific, MA5-24,844, 1:200), and CD68 (Bio-Rad, MCA1957, 1:200).

Techniques: Ex Vivo, Biomarker Discovery, Staining, Immunofluorescence, Control, Tissue, Muscles, Analysis

Journal: EJNMMI Research

Article Title: TSPO PET selectively detects the denervated skeletal muscle district remodeling after experimental peripheral nerve injury

doi: 10.1186/s13550-026-01497-7

Figure Lengend Snippet: Early structural remodeling and onset of denervation-induced muscle atrophy following sciatic nerve injury. ( A ) Representative immunofluorescence images of TSPO (green) signal in skeletal muscle cross-sections from tibialis muscle harvested from contralateral hindlimb (Control), sham-operated (Sham), and homolateral side of injured sciatic nerve crush (Crush). Nuclei are counterstained with DAPI (blue). Specimen downstream the nerve damage showed a marked increase in % area occupied by TSPO immunofluorescence ( B ). CD68 and TSPO immunostaining and their colocalization in the tibialis anterior muscle downstream the nerve damage. ( D ) Representative Western blot analysis of TSPO protein levels in tibialis anterior muscle homogenates one-week after surgery. Total protein staining was used for normalization. ( E ) Densitometric quantification of TSPO expression normalized to total protein. TSPO levels were significantly increased in denervated muscle compared with control and sham groups, whereas no difference was observed between control and sham mice. Data are presented as mean ± SD; each dot represents one biological replicate. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (***p < 0.001)

Article Snippet: Sections were then incubated overnight at 4 °C with primary antibodies against laminin α-2 (Santa Cruz Biotechnology, sc-59854, 1:200), TSPO (Thermo Fisher Scientific, MA5-24,844, 1:200), and CD68 (Bio-Rad, MCA1957, 1:200).

Techniques: Immunofluorescence, Control, Immunostaining, Western Blot, Analysis, Staining, Expressing

Journal: EJNMMI Research

Article Title: TSPO PET selectively detects the denervated skeletal muscle district remodeling after experimental peripheral nerve injury

doi: 10.1186/s13550-026-01497-7

Figure Lengend Snippet: Correlation between in vivo [18F]DPA-714 uptake and ex vivo markers of muscle inflammation and remodeling. Scatter plots showing the association between muscle [18F]DPA-714 uptake, expressed as SUV, and corresponding ex vivo tissue markers: CD68 immunoreactivity ( A ), TSPO immunofluorescence ( B ), TSPO protein expression by Western blot ( C ), and minimum Feret diameter (MFD) as an index of myofiber size ( D ). Each point represents an individual muscle sample normalized as % of the mean value observed in the corresponding experimental group. Injured/crush, contralateral/control, and sham muscles are shown with distinct colors. Solid lines indicate the best-fit linear regression. R 2 and p values are reported in each panel

Article Snippet: Sections were then incubated overnight at 4 °C with primary antibodies against laminin α-2 (Santa Cruz Biotechnology, sc-59854, 1:200), TSPO (Thermo Fisher Scientific, MA5-24,844, 1:200), and CD68 (Bio-Rad, MCA1957, 1:200).

Techniques: In Vivo, Ex Vivo, Tissue, Immunofluorescence, Expressing, Western Blot, Control, Muscles

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