anti p62  (Proteintech)

Journal: bioRxiv

Article Title: 3D Light Sheet Microscopy Reveals Aging Osteoarthritis-associated Joint-innervated Nerve Remodeling in Mouse Knee Joints

doi: 10.64898/2026.01.09.698640

Figure Lengend Snippet: Male mice experience more severe age-associated OA relative to female mice. Age associated changes in osteoarthritis severity, pain sensitivity and expression of CGRP and PIEZO2 in the DRGs. (A) Schematic of experimental design. (B) Representative sagittal sections stained with Safranin-O/Fast-Green. Blue arrows point degenerated articular cartilage. Scale bars, 200 µm. (C) OARSI scores of the articular cartilage (n = 5-7 mice/group). (D) Quantitative analysis of knee hyperalgesia using the Pressure application measurement (PAM) (n = 5-7 mice/group). (E) Representative immunofluorescent images of PGP9.5, CGRP and PIEZO2 staining in L3-L5 DRGs. Scale bar, 100 μm. (F) CGRP + neuron counts (n = 4-5 mice/group) and (G) anti-Piezo2 relative staining intensity (n = 4-5 mice/group) in DRG sections. Mean ± 95% CI. OARSI and PAM data were analyzed using a non-parametric Kruskal–Wallis test followed by Dunn’s multiple-comparison post hoc test. CGRP and Piezo2 data were analyzed using two-way ANOVA with sex and age as factors followed by uncorrected Fisher’s LSD post hoc tests.

Article Snippet: Given that CGRP is a secreted neuropeptide and is also present in vascular smooth muscle surrounding blood vessels, the complete nerve network was first reconstructed in 3D using PGP9.5+ filaments in Imaris (v10.1.1).

Techniques: Expressing, Staining, Comparison

Journal: bioRxiv

Article Title: 3D Light Sheet Microscopy Reveals Aging Osteoarthritis-associated Joint-innervated Nerve Remodeling in Mouse Knee Joints

doi: 10.64898/2026.01.09.698640

Figure Lengend Snippet: Age-associated increase in total knee joint innervation in male mice. (A) Representative three-dimensional (3D) images of knee joints labeled with anti-PGP9.5 (green) and the reconstructed µCT scans. Quantified regions are shown in yellow (x-y axis) and orange (y-z axis) boxes. (B) Quantification of the imaging coordinate length of the knee joint in young and aged male and female mice respectively in the x-axis, y-axis and z-depth. (C) Representative 3D anti-PGP9.5 (green) micrographs of the knee joint of My (young male), Ma (aged male), Fy (young female) and Fa (aged female); white line traces PGP9.5+ nerve filaments using Imaris v10.1.1 software. (D) Representative MATLAB-generated images showing the boundary volume (gray) encapsulating the PGP9.5+ segment coordinates (red) in My, Ma, Fy and Fa (E) Quantification of the total boundary volume, (F) PGP9.5+ filament length, (G) PGP9.5+ filament length/total boundary volume, (H) PGP9.5+ branch points, and (I) PGP9.5+ branch points/total filament length in young and aged male and female mice, respectively. N = 5 mice/group. Data were analyzed using two-way ANOVA to assess the effects of age and sex, followed by uncorrected Fisher’s LSD post hoc tests. Data are presented as mean ± 95%CI.

Article Snippet: Given that CGRP is a secreted neuropeptide and is also present in vascular smooth muscle surrounding blood vessels, the complete nerve network was first reconstructed in 3D using PGP9.5+ filaments in Imaris (v10.1.1).

Techniques: Labeling, Imaging, Software, Generated

Journal: bioRxiv

Article Title: 3D Light Sheet Microscopy Reveals Aging Osteoarthritis-associated Joint-innervated Nerve Remodeling in Mouse Knee Joints

doi: 10.64898/2026.01.09.698640

Figure Lengend Snippet: Change in CGRP⁺/PGP9.5⁺ nociceptive nerve innervation in the aging knee joints. (A) (top) Representative 3D Light-Sheet Micrographs of knee-innervating nerve fibers stained with anti-PGP9.5 (green) and anti-CGRP (red) in M y , M a , F y and F a ; CGRP + nerve fibers are traced using IMARIS. CGRP⁺ nerve innervation was quantified by first segmenting total nerve fibers using PGP9.5 immunolabeling. A single, fixed intensity threshold was then applied uniformly across all samples to identify CGRP⁺ signal within the PGP9.5⁺ nerve segments to quantify CGRP + PGP9.5 + nerve filament. (B) Total length of CGRP + PGP9.5 + nerve filament. (C) Total CGRP + PGP9.5 + nerve filament length/boundary volume. (D) Total branch points in CGRP + PGP9.5 + nerve filaments. (E) CGRP + PGP9.5 + branch points/total CGRP + PGP9.5 + filament length. N = 3 mice/group.

Article Snippet: Given that CGRP is a secreted neuropeptide and is also present in vascular smooth muscle surrounding blood vessels, the complete nerve network was first reconstructed in 3D using PGP9.5+ filaments in Imaris (v10.1.1).

Techniques: Staining, Immunolabeling

Journal: bioRxiv

Article Title: 3D Light Sheet Microscopy Reveals Aging Osteoarthritis-associated Joint-innervated Nerve Remodeling in Mouse Knee Joints

doi: 10.64898/2026.01.09.698640

Figure Lengend Snippet: Orthogonal planes of view (coronal, sagittal and transverse) from PGP9.5/CGRP immunolabeled knee joints from (A) young male and (B) aged male mice, and from PGP9.5/TH immunolabeled knee joints from (C) young male and (D) aged male mice, depicting the depth of nerve infiltration into the various tissues of the knee joint. Insets capture the femur (inset B), infrapatellar fat pad (inset C), and joint capsule/synovium (inset D). CGRP+ sensory nerves (panels A,B) and TH+ sympathetic nerves (panels C,D) appear to innervate the extrasynovial fat and joint capsule (insets C,D), whereas minimal staining is seen in the femoral bone (inset B), which likely reflects insufficient antibody penetration into bone. White insets are 300 μm x 300 μm.

Article Snippet: Given that CGRP is a secreted neuropeptide and is also present in vascular smooth muscle surrounding blood vessels, the complete nerve network was first reconstructed in 3D using PGP9.5+ filaments in Imaris (v10.1.1).

Techniques: Immunolabeling, Staining

Journal: bioRxiv

Article Title: 3D Light Sheet Microscopy Reveals Aging Osteoarthritis-associated Joint-innervated Nerve Remodeling in Mouse Knee Joints

doi: 10.64898/2026.01.09.698640

Figure Lengend Snippet: Spatial mapping of segment positions in the medial and lateral compartments of the knee joint in young and aged male mice (M y , M a ). (A) k-means clustering was performed in Matlab to partition the segment position data into medial and lateral clusters for PGP9.5, CGRP and TH innervation. The percentage of segments in medial and lateral clusters was quantified for PGP9.5 (n = 5 mice/group) (B) CGRP (n = 3 mice/group) (C), TH (n = 4 mice/group) (D). Medial-lateral proportions for individual samples are connected by lines, with bars representing the mean of each compartment. Repeated measures two-way ANOVA with post-hoc Fisher’s Least Significant Difference (LSD) test was performed to statistically compare differences in medial-lateral nerve proportions across young and aged male mice, matching compartments from individual mice.

Article Snippet: Given that CGRP is a secreted neuropeptide and is also present in vascular smooth muscle surrounding blood vessels, the complete nerve network was first reconstructed in 3D using PGP9.5+ filaments in Imaris (v10.1.1).

Techniques:

Journal: bioRxiv

Article Title: 3D Light Sheet Microscopy Reveals Aging Osteoarthritis-associated Joint-innervated Nerve Remodeling in Mouse Knee Joints

doi: 10.64898/2026.01.09.698640

Figure Lengend Snippet: Spatial mapping of segment positions in the medial and lateral compartments of the knee joint in aged female mice with osteoarthritis compared to young female mice. (A) k-means clustering was performed in Matlab to partition the segment position data into medial and lateral clusters for PGP9.5, CGRP and TH innervation. The percentage of segments in medial and lateral clusters was quantified for PGP9.5 (n = 5 mice/group) (B) CGRP (n = 3 mice/group) (C) TH (n = 4 mice/group) (D) Medial-lateral proportions for individual samples are connected by lines, with bars representing the mean of each compartment. Repeated measures two-way ANOVA with post-hoc Fisher’s Least Significant Difference (LSD) test was performed to statistically compare differences in medial-lateral nerve proportions across young and aged female mice, matching compartments from individual mice.

Article Snippet: Given that CGRP is a secreted neuropeptide and is also present in vascular smooth muscle surrounding blood vessels, the complete nerve network was first reconstructed in 3D using PGP9.5+ filaments in Imaris (v10.1.1).

Techniques:

Journal: Bone Research

Article Title: PTH induced osteoblast Slit3 to decrease aberrant sensory innervation in degenerated vertebral endplates to relieve low back pain in mice

doi: 10.1038/s41413-025-00488-z

Figure Lengend Snippet: PTH treatment alters innervation in the degenerated spine. a Representative images of PGP9.5-positive fibers (upper row, Green) and CGRP-positive fibers (lower row, Red) in the lumbar vertebral body and endplate of aged mice treated with PTH or Veh for 1 or 2 months. Scale bar: 100 µm. Quantitative analysis of the length of PGP9.5-positive fibers ( b ) or CGRP-positive fibers ( c ) in the lumbar vertebral body of aged mice treated with PTH or Veh for 1 month or 2 months ( n ≥ 5, t -test). Representative images ( d ) and quantitative mean immunofluorescent (IF) intensity ( e ) of CGRP-positive neurons in the dorsal root ganglia (DRG) (L1-L2) of aged mice treated with PTH or Veh for 1 month or 2 months. Scale bar: 100 µm. ( n ≥ 6, t -test). f Protein levels of CGRP in DRG tissue (L1–L2) relative to the expression of GAPDH in aged mice treated with PTH or Veh for 1 or 2 months, respectively. ( n = 5). Representative images and quantitative analysis of the length of PGP9.5-positive ( g , h , Green) and CGRP-positive ( g , i , Red) fibers in the vertebral body and endplate of WT young mice 2 months after LSI surgery and treated with PTH or Veh for 2 months. Scale bar: 100 µm. ( n ≥ 6, t -test). Representative images ( j ) and quantitative mean IF intensity ( k ) of CGRP-positive neurons in the DRG of WT young mice 2 months after LSI surgery and treated with PTH or Veh for 2 months. Scale bar: 100 µm. ( n ≥ 5, t -test). DAPI stains nuclei blue. EP endplate, VB vertebral body. * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001

Article Snippet: A range of primary antibodies was used for this purpose, including those specific for mouse β3 tubulin (1:500, 2G10, Thermo Fisher), CGRP (1:1 000, sc-57053, Santa Cruz), PGP9.5 (1:1 000, SAB4503057, Sigma), IB4 (1:1 000, I21441 , Thermo Fisher), TH (1:1 000, AB152, Sigma), Slit3 (1:1 000, AF3629, Biotechne), E47 (1:1 000, sc-416, Santa Cruz), FoxA2 (1:1 000, 22474-1-AP, Proteintech), and GAPDH (1:2 000, 14C10, Cell Signaling), which facilitated the determination of protein concentrations in the lysates.

Techniques: Expressing

Journal: Bone Research

Article Title: PTH induced osteoblast Slit3 to decrease aberrant sensory innervation in degenerated vertebral endplates to relieve low back pain in mice

doi: 10.1038/s41413-025-00488-z

Figure Lengend Snippet: Loss of PPR in osteoblasts ameliorates PTH response in endplate degeneration and pain behaviors during spine degeneration. BV/TV percentage ( a ), total porosity percentage ( b ), and total pore space ( c ) in PPR OCN −/− LSI mice with PTH or Veh treatment for 2 months. ( n = 5, t -test). Behavior evaluation of PPR OCN −/− LSI mice treated with PTH or Veh for 2 months included pressure tolerance of the lumbar spine region ( d , n ≥ 3, t -test), paw withdraw times of the hind paw response to 0.4 g Von Frey filament stimulation (10 times in total) ( e , n = 8, t -test), and latency of paw withdrawal post-thermal stimulation ( f , n ≥ 7, t -test). Representative images of PGP9.5 and CGRP-positive fibers ( g ), quantitative analysis of fiber length in the lumbar vertebral body of PPR OCN −/− LSI mice with PTH or Veh treatment ( h , i ). Scale bar: 100 µm. ( n ≥ 5, t -test). Representative images showing CGRP-positive neuron ( j ) and quantitative analysis of mean intensity of immunofluorescence ( k ) in the DRG of PPR OCN −/− LSI mice. Scale bar: 100 µm. ( n = 4, t -test). * P < 0.05, ** P < 0.01

Article Snippet: A range of primary antibodies was used for this purpose, including those specific for mouse β3 tubulin (1:500, 2G10, Thermo Fisher), CGRP (1:1 000, sc-57053, Santa Cruz), PGP9.5 (1:1 000, SAB4503057, Sigma), IB4 (1:1 000, I21441 , Thermo Fisher), TH (1:1 000, AB152, Sigma), Slit3 (1:1 000, AF3629, Biotechne), E47 (1:1 000, sc-416, Santa Cruz), FoxA2 (1:1 000, 22474-1-AP, Proteintech), and GAPDH (1:2 000, 14C10, Cell Signaling), which facilitated the determination of protein concentrations in the lysates.

Techniques: Immunofluorescence

Journal: Bone Research

Article Title: PTH induced osteoblast Slit3 to decrease aberrant sensory innervation in degenerated vertebral endplates to relieve low back pain in mice

doi: 10.1038/s41413-025-00488-z

Figure Lengend Snippet: Osteoblasts respond to PTH treatment with increased Slit3 transcription and translation. mRNA expression of the Slit3 in the endplate tissue of WT young mice treated with Veh and aged mice treated with Veh or PTH for 1 month ( a ), and WT LSI mice treated with veh or PTH for 2 months ( b ) ( n = 3, one-way ANOVA with Tukey’s multiple comparisons test). c mRNA expression of the Slit3 in MC3T3 cells cultured in osteoblast-stimulated medium and subsequently treated with Veh or PTH at different doses for 3 days ( n = 3, t -test). d , e Protein expression level of Slit3 in MC3T3 cells cultured in osteoblast-stimulated medium and treated with Veh (PBS) or PTH for 3 days. ( n = 3, t -test). Representative images ( f ) and quantification ( g ) of IF staining of PGP9.5 positive primary DRG neuron fibers crossing the microgroove barrier (microfluid assay) cultured in condition medium (CM), CM + PTH, CM + PTH plus mouse Slit3 antibody (1 µg/mL), or CM + Veh (1X PBS) plus human recombinant Slit3 (1.25 µg/mL) for 1 week. Representative images showing co-immunostaining for OCN (green) and Slit3 (red) in the lumbar spine sections and quantitative analysis of number of Slit3 + cells in trabecular bone (TB) and endplate (EP) of aged mice ( h – j ) and PPR OCN −/− LSI mice ( k – m ) treated with PTH or Veh for 2 months. Scale bar: 100 µm. ( n ≥ 5, t -test). * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001

Article Snippet: A range of primary antibodies was used for this purpose, including those specific for mouse β3 tubulin (1:500, 2G10, Thermo Fisher), CGRP (1:1 000, sc-57053, Santa Cruz), PGP9.5 (1:1 000, SAB4503057, Sigma), IB4 (1:1 000, I21441 , Thermo Fisher), TH (1:1 000, AB152, Sigma), Slit3 (1:1 000, AF3629, Biotechne), E47 (1:1 000, sc-416, Santa Cruz), FoxA2 (1:1 000, 22474-1-AP, Proteintech), and GAPDH (1:2 000, 14C10, Cell Signaling), which facilitated the determination of protein concentrations in the lysates.

Techniques: Expressing, Cell Culture, Staining, Recombinant, Immunostaining

Journal: Bone Research

Article Title: PTH induced osteoblast Slit3 to decrease aberrant sensory innervation in degenerated vertebral endplates to relieve low back pain in mice

doi: 10.1038/s41413-025-00488-z

Figure Lengend Snippet: Efficacy of PTH treatment is diminished in osteoblastic Slit3 knockout mice with spinal degeneration. Vertebral endplate bone structure analyses by micro-CT: bone volume per tissue volume (BV/TV) percentage ( a ), total porosity percentage ( b ), and total pore space ( c ) in Slit3 OCN −/− LSI mice with PTH (40 µg/kg/d) or Veh treatment for 2 months ( n = 7, t -test). Behavior evaluations included pressure tolerance of the lumbar spine assessed via the force threshold ( d ), total distance covered in spontaneous activity in 2 days ( e ), and latency of paw withdrawal post-thermal stimulation ( f ) for Slit3 OCN −/− LSI mice with PTH or Veh treatment for 2 months ( n = 7, t -test). g Western blot analysis of protein expression levels of β3 tubulin, CGRP, PGP9.5 relative to GAPDH in endplate tissue (L3-L5) in Slit3 OCN −/− LSI mice treated with PTH or Veh for 2 months. ( n = 5). Representative images of PGP9.5 (red) and CGRP (green)-positive fibers ( h ) and quantitative analysis of fiber length in the lumbar vertebral body of Slit3 OCN −/− LSI mice with PTH or Veh treatment ( i , j ) ( n = 5, t -tset). Scale bar: 100 µm. k Western blot analysis of protein expression levels of CGRP in DRG tissue (L1–L2), normalized to GAPDH expression, in Slit3 OCN −/− LSI mice treated with PTH or Veh for 2 months ( n = 5). Representative IF images showing CGRP-positive (green) neurons in DRG sections ( l ), followed by quantitative analysis of the mean IF intensity for CGRP ( m ) in the DRG of Slit3 OCN −/− LSI mice treated with PTH or Veh for 2 months ( n = 5, t -tset). Scale bar: 100 µm

Article Snippet: A range of primary antibodies was used for this purpose, including those specific for mouse β3 tubulin (1:500, 2G10, Thermo Fisher), CGRP (1:1 000, sc-57053, Santa Cruz), PGP9.5 (1:1 000, SAB4503057, Sigma), IB4 (1:1 000, I21441 , Thermo Fisher), TH (1:1 000, AB152, Sigma), Slit3 (1:1 000, AF3629, Biotechne), E47 (1:1 000, sc-416, Santa Cruz), FoxA2 (1:1 000, 22474-1-AP, Proteintech), and GAPDH (1:2 000, 14C10, Cell Signaling), which facilitated the determination of protein concentrations in the lysates.

Techniques: Knock-Out, Micro-CT, Activity Assay, Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Clusterin protects against HFpEF by inhibiting UCHL1-mediated NLRP3 deubiquitylation and inflammasome activation

doi: 10.3389/fphar.2025.1704023

Figure Lengend Snippet: CLU interacted with UCHL1 and promoted NLRP3 degradation. (A) Western blot analysis of the interaction between CLU and UCHL1 detected by co-immunoprecipitation (Co-IP). (B) Western blot analysis of CLU, LC3B, p62, UCHL1, and NLRP3 in cells with or without CLU overexpression. β-actin was used as a loading control. Quantification of protein expression for (C) NLRP3 and (D) UCHL1. (E) The ubiquitination of NLRP3 promoted by CLU. (F) The ubiquitination of NLRP3 inhibited by UCHL1. (G) Western blot analysis of NLRP3 degradation after cycloheximide (CHX) treatment in CLU overexpressing cells. (H) Quantitative analysis of NLRP3 degradation.

Article Snippet: Protein samples were incubated with anti-CLU (Cell Signaling Technology, Cat# 34642), anti-NLRP3 (Proteintech, Cat# 68102-1-Ig), anti-Col1a2 (Abcam, Cat# ab308455), anti-UCHL1 (Proteintech, Cat# 14730-1-AP), anti-p62 (Proteintech, Cat# 18420-1-AP), anti-LC3B (Proteintech, Cat# 14600-1-AP).

Techniques: Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Over Expression, Control, Expressing, Ubiquitin Proteomics