Journal: The Journal of Physiological Sciences : JPS

Article Title: Genetic inactivation of TRPM4 does not alter the temperature-dependent movement of mouse microglia

doi: 10.1016/j.jphyss.2026.100067

Figure Lengend Snippet: Generation of Trpm4 -knockout mouse line. A. Map of the mouse Trpm4 locus showing exons 15 and 16 targeted for deletion by sgRNAs. Top, WT allele. Bottom, KO allele. The sgRNAs were designed to target introns 14 and 16 flanking exons 15 and 16. Specific primers for amplifying the WT(Fw2/Rv) or KO (Fw1/Rv) allele were used. B. A representative result of genotyping PCR using a mixture of the Fw1, Fw2, and Rv primers. Two fragments were amplified from heterozygote mouse samples, each indicating the WT or KO fragment. The bottom fragment (695 bp) was amplified using the WT primer pair. The top fragment (928 bp) was amplified using the KO primer pair, indicating successful deletion of exons 15 and 16, which was confirmed by direct sequencing. (C and D). The expression patterns of Trpm4 gene in mouse brain (C) or primary microglia (D) using Gapdh as a control. In (C), “M4-plasmid” indicates an amplified fragment using mouse TRPM4 plasmid as a template. E. Western blot analysis of Trpm4 in mouse TRPM4-expressing HEK293T cells (M4-HEK) and mouse colon samples from WT or TRPM4KO mice. The expected molecular weight of Trpm4 is 134 kDa.

Article Snippet: After transferring the proteins to a nitrocellulose membrane, the membrane was blocked for 1 h at room temperature and then incubated with anti-TRPM4 antibody (1:300, ACC044, Alomone) overnight at 4 °C.

Techniques: Knock-Out, Amplification, Sequencing, Expressing, Control, Plasmid Preparation, Western Blot, Molecular Weight

Journal: The Journal of Physiological Sciences : JPS

Article Title: Genetic inactivation of TRPM4 does not alter the temperature-dependent movement of mouse microglia

doi: 10.1016/j.jphyss.2026.100067

Figure Lengend Snippet: Genetic elimination of Trpm4 does not alter the temperature-dependent microglia movement. A. Trajectories of primary microglia from a representative preparation of WT and TRPM4KO mice recorded for 2 h at 33 °C, 37 °C, and 40 °C. Paths are arranged to show origins at x (horizontal axis) = y (vertical axis) = 0. Each line indicates the trajectory of a single cell. n indicates the number of cells analyzed per preparation. B. The average distances of migrating microglia isolated from WT or TRPM4KO mice exposed to 33 °C (WT, n = 117; TRPM4KO, n = 151), 37 °C (WT, n = 235; TRPM4KO, n = 240), or 40 °C (WT, n = 150; TRPM4KO, n = 175) were measured. Open circles indicate the migration distance of each microglia over 2 h. Horizontal lines indicate means ± SEM. At 33 °C, 37 °C, and 40 °C, WT microglia moved 112.94 ± 6.1 μm, 175.28 ± 5.24 μm, and 204.31 ± 7.27 μm, respectively, whereas TRPM4KO microglia moved 113.46 ± 5.1 μm, 175.28 ± 4.13 μm, and 201.35 ± 5.61 μm, respectively. **P < 0.01 (two-way ANOVA followed by post hoc Bonferroni test for multiple comparisons).

Article Snippet: After transferring the proteins to a nitrocellulose membrane, the membrane was blocked for 1 h at room temperature and then incubated with anti-TRPM4 antibody (1:300, ACC044, Alomone) overnight at 4 °C.

Techniques: Single Cell, Isolation, Migration

Journal: Biomaterials Research

Article Title: Injectable Poloxamer and Hyaluronic Acid Hydrogel for Sustained Co-Delivery of Dexamethasone and Lidocaine Ameliorates Neuropathic Pain

doi: 10.34133/bmr.0373

Figure Lengend Snippet: TRPV1-mediated nociceptive sensitization in the DRG and spinal cord dorsal horn (SC) following CCI and treatment. (A) Schematic illustration of the proposed mechanism: Injury-induced TRPV1 ion channel activation triggers calcium influx and downstream CGRP release, which activates adenylate cyclase/PKA signaling to amplify neuropathic pain sensitization. (B) Representative immunofluorescence images of the DRG stained for TRPV1 (red), NeuN (green), and DAPI (blue). The Injury group shows marked up-regulation of TRPV1 in sensory neurons. Dex/Lid@PLX/HA treatment substantially reduces TRPV1 expression, restoring it to near-Naive levels. Scale bars, 200 μm (overview) and 50 μm (inset). (C) Immunofluorescence staining for TRPV1 (red) and NeuN (green) in the spinal dorsal horn. The dashed line indicates the dorsal horn boundary. Dex/Lid@PLX/HA significantly suppresses injury-induced central TRPV1 up-regulation. Scale bar, 200 μm. (D to F) Quantitative analysis of the relative TRPV1 + area in the DRG (top) and spinal cord (middle), and the SGC/neuron ratio. Dex/Lid@PLX/HA shows marked suppression of TRPV1 overexpression relative to the injury group. Data are presented as mean ± SEM. **** P < 0.0001, ** P < 0.01, * P < 0.05, ns: not significant.

Article Snippet: The sections were then incubated overnight at 4 °C with the following primary antibodies: TRPV1 (Alomone Labs, catalog number ACC-030-GP), Iba-1 (Abcam, catalog number ab5076), NeuN (Abcam, catalog number ab104224), CD68 (Abcam, catalog number ab31630), CD163 (Abcam, catalog number ab182422), CGRP (Abcam, catalog number ab47027), GFAP (Millipore, catalog number MAB360), and NF200 (Abcam, catalog number ab8135).

Techniques: Activation Assay, Immunofluorescence, Staining, Expressing, Over Expression

Journal: bioRxiv

Article Title: A novel role for TRPV1 in macrophage giant cell formation

doi: 10.64898/2026.05.11.724406

Figure Lengend Snippet: (A) Immunoblot analysis shows TRPV1, TRPA1, and actin levels in WT BMDMs 48 h following treatment with or without IL-4 plus GMCSF (25 ng/ml). (B-C) Densitometric quantification of immunoblot data from (A) (n = 3 biological replicates; one-way ANOVA, **p < 0.01, ***p < 0.001). (D) Representative immunofluorescence images of WT BMDMs stained for TRPV1 (red) using anti-TRPV1 IgG (original magnification, 60 x; scale bar, 2 μm; n = 10 cells per condition). Statistical analysis by Student’s t-test, ***p < 0.001.

Article Snippet: Primary antibodies comprised anti-actin (cat# 4970S; Cell Signaling Technology, Danvers, MA) and anti-TRPV1 (cat# ACC-030), anti-TRPV2 (cat# ACC-039), and anti-TRPA1 (cat# ACC-037) from Alomone Labs. Species-appropriate secondary antibodies (goat, rabbit, and mouse) were obtained from Jackson ImmunoResearch.

Techniques: Western Blot, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: A novel role for TRPV1 in macrophage giant cell formation

doi: 10.64898/2026.05.11.724406

Figure Lengend Snippet: (A) Representative Giemsa-stained images of multinucleated FBGCs in WT BMDMs left untreated or stimulated with IL-4 plus GMCSF (25 ng/ml, 96 h), in the presence or absence of the TRPV1 antagonist AMG. (B-D) Quantitative analysis of FBGC formation from (A): (B) number of FBGCs per high-power field, (C) percentage of fused BMDMs, and (D) average FBGC size. Data represent n = 3 biological replicates with 5 images per group; scale bar, 100 μm; Student’s t-test, ***p < 0.001, ****p < 0.0001. (E) Representative immunofluorescence images of WT BMDMs transfected with scramble or TRPV1-targeting siRNA, stained for TRPV1 (red) using anti-TRPV1 IgG (original magnification, 60x; scale bar, 2 μm). (F) Quantification of TRPV1 fluorescence intensity (n = 10 cells per condition; Student’s t-test, ***p < 0.001). (G) Immunoblot showing TRPV1 expression in WT BMDMs 48 h after transfection with scramble or TRPV1 siRNA. (H-J) Quantification of FBGC formation following TRPV1 knockdown: (H) number of FBGCs per high-power field, (I) percentage of fused BMDMs, and (J) average FBGC size. Data represent n = 3 biological replicates with 5 images per group; Student’s t-test, ***p < 0.001.

Article Snippet: Primary antibodies comprised anti-actin (cat# 4970S; Cell Signaling Technology, Danvers, MA) and anti-TRPV1 (cat# ACC-030), anti-TRPV2 (cat# ACC-039), and anti-TRPA1 (cat# ACC-037) from Alomone Labs. Species-appropriate secondary antibodies (goat, rabbit, and mouse) were obtained from Jackson ImmunoResearch.

Techniques: Staining, Immunofluorescence, Transfection, Fluorescence, Western Blot, Expressing, Knockdown

Journal: bioRxiv

Article Title: Microglial and Neuronal Cross-talk in the Nucleus Accumbens

doi: 10.64898/2026.05.01.722235

Figure Lengend Snippet: (A) Immunohistochemical panel showing that ATP P2X4 receptors are on microglia. First image depicts microglia, second shows P2X4 receptor expression with third image showing merge, arrows indicating microglia with P2X4 expression. (B) Immunohistochemical panel showing that ATP P2Y6 receptors are on microglia. First image depicts microglia, second shows P2Y6 receptor expression with third image showing merge, arrow indicates microglia with P2Y6 expression with inset showing zoomed in image. (C) Immunohistochemical panel showing that microglia have both D1 and D2 receptors. (D) Immunohistochemical panel showing that TRPM2 are expressed on microglia. First image depicts microglia, second shows TRPM2 receptor expression with third image showing merge, arrows indicating microglia with TRPM2 expression.

Article Snippet: Additional antibodies used include P 2 Y 6 receptors, P 2 X 4 receptors, D1 receptors, D2 receptors, and TRPM2 (Alomone Labs, Cat. No. ACC-043).

Techniques: Immunohistochemical staining, Expressing

Journal: bioRxiv

Article Title: Nanoscale CaV channel reorganization links α-synuclein pathology to calcium-dependent transcriptional dysregulation

doi: 10.64898/2026.05.01.719272

Figure Lengend Snippet: A Schematic illustrating imaging at the plasma membrane (PM). B Left: representative single-plane Airyscan confocal images of the PM showing Ca V 1.2 immunolabeling in control (CTL, black) and PFF-treated (red) neurons. Inset: MAP2 (pink) neuronal marker. Right: quantification of Ca V 1.2 cluster size, cluster density, and mean gray value (MGV) in the soma (a.) and dendrites (b.) of CTL (black) and PFF-treated (red) neurons. Dendritic measurements are shown separately for excitatory (dark blue) and inhibitory (light blue) populations. n = 20 somata per condition; n = 20 dendrites per group (CTL excitatory, CTL inhibitory, PFF excitatory, PFF inhibitory); two independent isolations with each isolation containing 8-10 pups. C Left: representative super-resolution TIRF localization maps showing Ca V 1.2 immunolabeling in CTL (black) and PFF-treated (red) neurons. Right: quantification of PM Ca V 1.2 cluster size, cluster density, and nearest-neighbor distance in the somatic region. n = 16 neurons per condition; two independent isolations. D Same experimental design as in ( B ), with neurons immunolabeled for Ca V 2.1. n = 19 (CTL) and n = 20 (PFF) somata; n = 20 dendrites per group (CTL excitatory, CTL inhibitory, PFF excitatory, PFF inhibitory); two independent isolations. E Same experimental design as in ( C ), with neurons immunolabeled for Ca V 2.1. n = 16 neurons per condition; two independent isolations. Error bars represent SEM. Statistical significance was determined using two-tailed Mann-Whitney or unpaired two-tailed t-tests. ns, not significant; *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001. CTL, control; PFF, α-synuclein pre-formed fibril treatment.

Article Snippet: The following combinations were used: K V 2.1 (NeuroMab, K89/34) with Ca V 1.2 (Alomone, ACC-003); K V 2.1 (NeuroMab, K89/34) with Ca V 2.1 (Alomone, ACC-001); and K V 2.1 (NeuroMab, K89/34) with K V 2.1 (NeuroMab, Drk1).

Techniques: Imaging, Clinical Proteomics, Membrane, Immunolabeling, Control, Marker, Isolation, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Nanoscale CaV channel reorganization links α-synuclein pathology to calcium-dependent transcriptional dysregulation

doi: 10.64898/2026.05.01.719272

Figure Lengend Snippet: A Schematic representation of the CCAD peptide mechanism of action. B Left: representative single-plane Airyscan confocal images of the PM in CTL and PFF-treated neurons co-incubated with SCRBL or CCAD peptides and co-immunolabeled for Ca V 1.2 and K V 2.1. Conditions are shown as CTL;SCRBL (black), PFF;SCRBL (red), CTL;CCAD (gray), and PFF;CCAD (yellow). Right: quantification of Ca V 1.2 cluster size, K V 2.1 cluster size, and Ca V 1.2-K V 2.1 overlap area in the somatic region. n = 19 (CTL;SCRBL), n = 19 (PFF;SCRBL), n = 20 (CTL;CCAD), and n = 20 (PFF;CCAD) neurons; two independent isolations. C Schematic representation of the proximity ligation assay (PLA). D Top: representative Airyscan confocal PLA images showing Ca V 1.2-K V 2.1 proximity. Images are maximum intensity projections from Z-stacks spanning whole cells. Color coding as in ( B ). Bottom: quantification of PLA puncta density. n = 18 neurons per condition; two independent isolations. E Same experimental design as in ( D ), but assessing Ca V 2.1-K V 2.1 proximity. n = 18 neurons per condition; two independent isolations. Error bars represent SEM. Statistical significance in panels ( B, D-E ) was determined using two-way ANOVA with appropriate post hoc tests. ns, not significant; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. CTL, control; PFF, α-synuclein pre-formed fibril treatment; CCAD, calcium channel association domain peptide; SCRBL, scrambled control peptide.

Article Snippet: The following combinations were used: K V 2.1 (NeuroMab, K89/34) with Ca V 1.2 (Alomone, ACC-003); K V 2.1 (NeuroMab, K89/34) with Ca V 2.1 (Alomone, ACC-001); and K V 2.1 (NeuroMab, K89/34) with K V 2.1 (NeuroMab, Drk1).

Techniques: Incubation, Immunolabeling, Proximity Ligation Assay, Control

Journal: bioRxiv

Article Title: Nanoscale CaV channel reorganization links α-synuclein pathology to calcium-dependent transcriptional dysregulation

doi: 10.64898/2026.05.01.719272

Figure Lengend Snippet: A Left: representative FV4000 confocal images showing co-immunolabeling of CDK5 and K V 2.1 at the PM in control (CTL, black) and PFF-treated (red) neurons. Images are maximum intensity projections from three optical sections acquired at the PM. Right: quantification of CDK5-K V 2.1 overlap area and CDK5 puncta density in the somatic region. n = 18 (CTL) and n = 19 (PFF) neurons; two independent isolations. B Schematic representation of the roscovitine mechanism of action. C Top: representative FV4000 confocal images of the PM in CTL and PFF-treated neurons incubated with or without roscovitine and immunolabeled for pS603-K V 2.1. Conditions are shown as CTL (black), PFF (red), CTL;Rosco (blue), and PFF;Rosco (purple). Images are maximum intensity projections from Z-stacks spanning whole cells. Bottom: quantification of somatic pS603-Kv2.1 occupancy (% of soma area), cluster MGV, and cluster density. n = 19 (CTL), n = 20 (PFF), n = 20 (CTL;Rosco), and n = 20 (PFF;Rosco) neurons; two independent isolations. D Left: representative FV4000 confocal PLA images showing Ca V 1.2-K V 2.1 proximity. Images are maximum intensity projections from Z-stacks spanning whole cells. Color coding as in ( C ). Right: quantification of PLA puncta density. n = 20 neurons per condition; two independent isolations. E Same experimental design as in ( D ), but assessing Ca V 2.1-K V 2.1 proximity. n = 20 (CTL), n = 20 (PFF), n = 21 (CTL;Rosco), and n = 20 (PFF;Rosco) neurons; two independent isolations. Error bars represent SEM. Statistical significance in panel ( A ) was determined using two-tailed Mann-Whitney test; panels ( C – E ) were analyzed using two-way ANOVA with appropriate post hoc tests. ns, not significant; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. CTL, control; PFF, α-synuclein pre-formed fibril treatment; Rosco, roscovitine; pS603-K V 2.1, K V 2.1 phosphorylated at serine 603.

Article Snippet: The following combinations were used: K V 2.1 (NeuroMab, K89/34) with Ca V 1.2 (Alomone, ACC-003); K V 2.1 (NeuroMab, K89/34) with Ca V 2.1 (Alomone, ACC-001); and K V 2.1 (NeuroMab, K89/34) with K V 2.1 (NeuroMab, Drk1).

Techniques: Immunolabeling, Control, Incubation, Two Tailed Test, MANN-WHITNEY