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