Journal: International Journal of Nanomedicine

Article Title: Human serum albumin-based probes for molecular targeting of macrophage scavenger receptors

doi: 10.2147/IJN.S197990

Figure Lengend Snippet: Evaluation of Mal-HSA-FITC uptake by different cell types. Fluorescence microscopy images of Mal-HSA-FITC probes (green) incubated for 3 h with ( A ) THP-1 macrophages, ( C ) HUVECs (EC), and ( D ) HCtSMCs (SMC). ( B ) Mal-HSA-FITC uptake by different cell types plotted against time. The results represent data from technical triplicates (9 images/well and timepoint) and are presented as mean and SEM. Data show significantly increased uptake of modified HSA probes by THP-1 cells compared with HUVEC (**** P <0.0001) and HCtSMCs ( #### P<0.0001) at 2.5 h and 3 h. None of the cell types recognized HSA-FITC (data not shown) (repeated measures two-way ANOVA (factors: cell type and time) and Tukey’s multiple comparison test). Abbreviations: HSA, human serum albumin; GCU, green calibration unit.

Article Snippet: Human carotid smooth muscle cells (HCtSMCs) (Cell Applications, San Diego, CA, USA) were cultured up to passage 7 in human smooth muscle cell media (Cell Applications).

Techniques: Fluorescence, Microscopy, Incubation, Modification, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: Extracellular calcium increases CXCR4 expression on bone marrow-derived cells and enhances pro-angiogenesis therapy

doi: 10.1111/j.1582-4934.2009.00691.x

Figure Lengend Snippet: Internalization of CXCR4 after binding with SDF-1. BMC were incubated with PBS with or without calcium for 4 hrs and then mixed with SDF-1 for 1 hr at 37° C. The surface (A) and intracellular (B) CXCR4 were measured by FACS. Surface CXCR4 was decreased and intracellular CXCR4 was increase after binding with SDF-1. * P < 0.05.

Article Snippet: BMCs (1 × 10 6 cells) were incubated with 0.5 mM CaCl 2 in PBS at 37°C for 4 hrs, then washed with PBS, re-suspended in 200 μl PBS containing recombinant mouse SDF-1α (final concentration 500 ng/ml, R&D Systems), and incubated at 37°C for 2 hr to allow SDF-1 to bind to the CXCR4 and the internalization of CXCR/DF-1 complex.

Techniques: Binding Assay, Incubation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Extracellular calcium increases CXCR4 expression on bone marrow-derived cells and enhances pro-angiogenesis therapy

doi: 10.1111/j.1582-4934.2009.00691.x

Figure Lengend Snippet: BMC migration towards SDF-1. (A) BMCs were pre-incubated with PBS with or without CaCl 2 for 4 hrs at 37°C and transferred to the upper chamber of inserts in a 24-well plate containing DMEM and 100 ng/ml SDF-1. Migrated cells were counted after 6 hrs incubation at 37°C. Inhibitors AMD3100, LY294002, L-NMMA and anti-CXCR4 antibody were added to the upper chamber along with BMCs. * P < 0.05 versus Calcium treated group; # P < 0.05 versus other groups. (B) Effect of the inhibitors on CXCR4 surface expression. BMC were incubated at 37°C for 4 hrs in PBS without or with 0.5 mM CaCl 2 plus specified inhibitors, and subjected same FACS analysis as in Fig. . Amount of surface CXCR4 on BMC was expressed as MFI.

Article Snippet: BMCs (1 × 10 6 cells) were incubated with 0.5 mM CaCl 2 in PBS at 37°C for 4 hrs, then washed with PBS, re-suspended in 200 μl PBS containing recombinant mouse SDF-1α (final concentration 500 ng/ml, R&D Systems), and incubated at 37°C for 2 hr to allow SDF-1 to bind to the CXCR4 and the internalization of CXCR/DF-1 complex.

Techniques: Migration, Incubation, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Extracellular calcium increases CXCR4 expression on bone marrow-derived cells and enhances pro-angiogenesis therapy

doi: 10.1111/j.1582-4934.2009.00691.x

Figure Lengend Snippet: Homing of injected BMC in ischemic site. BMCs from GFP mice were incubated in PBS with or without CaCl 2 at 37°C for 4 hrs, and then intravenously injected into WT BL6 mice with ischemic limb. SDF-1 protein (100 ng) was injected into the ischemic hindlimb muscle twice at consecutive days after the surgery. The hindlimb muscles were recovered 7 days after the cell injection, stained with DAPI for nucleus (blue), and examined for GFP cells incorporation under fluorescent microscopy. (A) Untreated BMC, (B) Ca-treated BMC, (C) BMC + SDF-1, (D) BMC/Ca + SDF-1. (E) The incorperated GFP cells were quantified as cells per high power field (HPF). * P < 0.05, and ** P < 0.01 versus all other groups ( n = 6).

Article Snippet: BMCs (1 × 10 6 cells) were incubated with 0.5 mM CaCl 2 in PBS at 37°C for 4 hrs, then washed with PBS, re-suspended in 200 μl PBS containing recombinant mouse SDF-1α (final concentration 500 ng/ml, R&D Systems), and incubated at 37°C for 2 hr to allow SDF-1 to bind to the CXCR4 and the internalization of CXCR/DF-1 complex.

Techniques: Injection, Incubation, Muscles, Staining, Microscopy

Journal: Journal of Cellular and Molecular Medicine

Article Title: Extracellular calcium increases CXCR4 expression on bone marrow-derived cells and enhances pro-angiogenesis therapy

doi: 10.1111/j.1582-4934.2009.00691.x

Figure Lengend Snippet: Effect of BMC injection on angiogenesis. BL6 mice with ischemia limb were injected intravenously with BM cells that were treated with or without CaCl 2 . SDF-1 protein was injected into the ischemic hindlimb muscle after the surgery. Blood flow of the lower limbs was measured using an laser Doppler perfusion images analyser. (A). Representative laser Doppler perfusion images at day 0 and day 21 after ischemia and cell injection. (B) Capillaries in the ischemic muscle from the mice at day 21 were identified by alkaline phosphatase cryosectional staining. (C). CD34 immunostaining of paraffin sections obtained from the ischemic muscle of mice at day 7. Brown spots are CD34 + cells (arrow). Blue represents cells counter-stained with haematoxylin. (D). Quantitative measurement of perfusion ratio of ischemic limbs to that of normal limbs at day 21 ( n = 6). (E) Quantification of capillary density on the tissue section, which is presented as the ratio of the number of capillaries to the number of muscle fibres. (F), Quantification of CD34 + cells around each muscle fibre. * P < 0.05 versus BMC/Ca group. # P < 0.05 versus other groups.

Article Snippet: BMCs (1 × 10 6 cells) were incubated with 0.5 mM CaCl 2 in PBS at 37°C for 4 hrs, then washed with PBS, re-suspended in 200 μl PBS containing recombinant mouse SDF-1α (final concentration 500 ng/ml, R&D Systems), and incubated at 37°C for 2 hr to allow SDF-1 to bind to the CXCR4 and the internalization of CXCR/DF-1 complex.

Techniques: Injection, Staining, Immunostaining

Journal: Journal of Neuroinflammation

Article Title: Actions of the antihistaminergic clemastine on presymptomatic SOD1-G93A mice ameliorate ALS disease progression

doi: 10.1186/s12974-016-0658-8

Figure Lengend Snippet: Clemastine short treatment affects the spinal cord pathology in SOD1-G93A mice at symptomatic phase of the disease. a Schematic representation of experimental design. b Spinal cord sections (L3–L5) from WT (~120 days) and vehicle- or clemastine-treated SOD1-G93A mice at PND 120 ( n = 4/group) are stained with cresyl violet (scale bar 100 μm) and assessment of motoneuron number is performed by percentage quantification ( n = 4/group) ( t test referred to WT * p < 0.05 or to vehicle-SOD1-G93A, # p < 0.05). c Spinal cord sections of vehicle- or clemastine-treated SOD1-G93A mice at PND 120 were then stained with anti-NeuN (scale bar = 40 μm). d Equal amounts of total lumbar spinal cord lysates from WT mice (~120 days) and vehicle- or clemastine-treated SOD1-G93A mice at PND 120 (n = 3/group) are subjected to western blotting with anti-Iba1, anti-CD68, anti-ARG-1, anti-CD163 and anti-GAPDH for protein normalisation. Data represent mean ± S.E.M. Statistical significance is calculated by student’s t test referred to WT, * p < 0.05, or to vehicle SOD1-G93A mice, # p < 0.05. e Equal amounts of lumbar spinal cord lysates from vehicle- or clemastine-treated SOD1-G93A mice at PND 120 ( n = 3/group) are subjected to western blotting with anti-P2X7, anti-P2X4, anti-P2Y12 and anti-GAPDH. Data represent mean ± S.E.M. Statistical significance was calculated by student’s t test, as referred to vehicle-treated SOD1-G93A mice, * p < 0.05

Article Snippet: After saturation with 5 % non-fat dry milk, blots were probed overnight at 4 °C with the specific primary antibodies: rabbit anti-ARG1 (1:1000, Millipore, USA), rat anti-CD68 (1:500, AbD Serotech, UK), rabbit anti-CD163 (1:100, Santa Cruz Biotechnology, USA), rabbit anti-Iba1 (1:1000, Wako, USA), rabbit anti-LC3B (1:1000, Cell Signaling Technology Inc, USA), rabbit anti-mTOR and phospho-mTOR (1:1000, Cell Signaling Technology Inc), rabbit anti-nuclear factor-kappa B (NF-kB) (1:500, Cell Signaling Technology Inc), rabbit anti-P2X4 (1:500, Alomone Labs, Israel), rabbit anti-P2X7 (1:500, Alomone Labs), rabbit anti-P2Y12 (1:200, Anaspec, USA), anti-p44/42 MAPK (ERK1/2) (L34F12) mouse antibody (1:1000) and anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1000, Cell Signaling Technology Inc.), rabbit anti-SOD1 (1:1000, Enzo Life sciences, USA), mouse anti-SQSTM1/p62 (1:1000, Abcam, USA).

Techniques: Staining, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Actions of the antihistaminergic clemastine on presymptomatic SOD1-G93A mice ameliorate ALS disease progression

doi: 10.1186/s12974-016-0658-8

Figure Lengend Snippet: Clemastine modulates the activation pattern of SOD1-G93A spinal microglia cultures from symptomatic mice. Spinal primary microglia from SOD1-G93A mice at PND 120 are exposed to clemastine 30 μM for 6 h. a Equal amounts of total lysates are subjected to western blotting with anti-CD68, anti-ARG-1, anti-CD163, anti-P2X7, anti-P2X4, anti-P2Y12 and anti-GAPDH for protein normalisation. Data represent mean ± S.E.M. of n = 3 independent experiments. Statistical significance is calculated by student’s t test referred to untreated cells ( Ctrl ), * p < 0.05. Cells are then analysed by means of a confocal microscope (scale bar 20 μm) after staining with anti-CD11b ( red ) and in b with anti-CD68 ( green ), in c with anti-ARG-1 ( green ), in d with anti-CD163 ( green ) and in e with anti-P2Y12 ( green )

Article Snippet: After saturation with 5 % non-fat dry milk, blots were probed overnight at 4 °C with the specific primary antibodies: rabbit anti-ARG1 (1:1000, Millipore, USA), rat anti-CD68 (1:500, AbD Serotech, UK), rabbit anti-CD163 (1:100, Santa Cruz Biotechnology, USA), rabbit anti-Iba1 (1:1000, Wako, USA), rabbit anti-LC3B (1:1000, Cell Signaling Technology Inc, USA), rabbit anti-mTOR and phospho-mTOR (1:1000, Cell Signaling Technology Inc), rabbit anti-nuclear factor-kappa B (NF-kB) (1:500, Cell Signaling Technology Inc), rabbit anti-P2X4 (1:500, Alomone Labs, Israel), rabbit anti-P2X7 (1:500, Alomone Labs), rabbit anti-P2Y12 (1:200, Anaspec, USA), anti-p44/42 MAPK (ERK1/2) (L34F12) mouse antibody (1:1000) and anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1000, Cell Signaling Technology Inc.), rabbit anti-SOD1 (1:1000, Enzo Life sciences, USA), mouse anti-SQSTM1/p62 (1:1000, Abcam, USA).

Techniques: Activation Assay, Western Blot, Microscopy, Staining

Journal: Cell reports

Article Title: Microglia are Indispensable for Synaptic Plasticity in the Spinal Dorsal Horn and Chronic Pain

doi: 10.1016/j.celrep.2019.05.087

Figure Lengend Snippet: (A) Spinal LTP at C-fiber synapses was induced by HFS at 10 and 20 V but not at 1V. The representative traces of C-fiber evoked fEPSPs were recorded before (i) and 3 h (ii) after high frequency stimulation (HFS) of the sciatic nerve at different intensities. Amplitude of C-fiber evoked fEPSPs (red vertical line) was determined automatically by parameter extraction software of WinLTP. Scale bars: x =100 ms, y = 0.2 mV. Summary data of the C-fiber responses expressed as mean ± SEM plotted vs. time were shown below (n = 6 mice/group). The arrow indicates the time point at which HFS was delivered. (B, C) Representative images and summary data show the expression of ATF3 in L4 DRG neurons 7 days after HFS at different intensities and 7 days after spared nerve injury (SNI). Scale bar, 100 μm. n = 3–4 mice/group, 3 slices/mouse, ***P < 0.001, compared with sham group, ###P < 0.001 vs. 20V HFS, one-way ANOVA with Tukey’s test. (D) The latencies to fall in rotarod test in different groups at 7 days after HFS at different intensities or SNI are shown. n = 5–12 mice for each group, ***P < 0.001, compared with sham group, one-way ANOVA with Tukey’s test. (E) Immunofluorescence of Iba1 (a marker for resident macrophage) and NIMP-R14 (a marker for neutrophil) in the stimulated sciatic nerve from different groups at 7days after HFS or SNI are shown (n = 3 mice/group, 2–3 sections/mouse). Scale bar: 50 μm. (F) Quantification of Iba1 expression in the sciatic nerve. ***P < 0.001, compared with sham group, ###P < 0.001 vs. 20V HFS, one-way ANOVA with Tukey’s test. (G, H) HFS at 10V and 20V significantly decreased 50% paw withdrawal threshold (PWT, G) to von Frey filaments and paw withdrawal latency (PWL, H) to radiant thermal stimuli, compared with the sham group or 1V HFS (n = 12 mice for 10V HFS group, n = 5–6 mice for other groups, **P < 0.01, ***P < 0.001 vs. sham group, two-way ANOVA with Fisher’s LSD test). (I) The decrease in mechanical thresholds induced by 10 V HFS was prevented by local application of 2% lidocaine (50 μl) at the stimulated sciatic nerve 15 min before HFS (n = 5–7 mice/group). ###P < 0.001 vs. HFS group, two-way ANOVA with Fisher LSD’s test. (J) Intrathecal injection of NMDA receptor antagonist D-AP5 (50 μg/ml, 5 μl) but not vehicle (Vehi, PBS) 30 min before HFS abolished HFS-induced mechanical hypersensitivity (n = 6–8 mice/group). ##P < 0.01, ###P < 0.001 vs. vehicle group, two-way ANOVA with Fisher LSD’s test.

Article Snippet: The tissue sections/cultured DRG neurons were first blocked with 5% donkey serum in 0.3% Triton X-100 (Sigma) for 60/30 min at room temperature (RT), and then incubated overnight at 4°C with a mixture of rabbit anti-ATF3 (1:500, sc-188, Santa Cruz Biotechnology, USA), mouse anti-CGRP antibody [4901] (1:2000, ab81887, Abcam, USA), isolectin B4-FITC (1:100, L2895, Sigma, USA), rabbit anti-GAP43 antibody (1:500, ab128005, Abcam, USA), rabbit anti-Substance P receptor antibody (1:500, AB5060, Millipore, USA.), rabbit anti-Iba1 (1:2000, 019–19741, Wako), goat anti-M-CSF antibody (1:500, AF416, R&D Systems, USA); rat anti-CD11b (1:500, 101202, Biolegend, USA); goat anti-Iba1 (1:500, ab5076, Abcam, USA) or rabbit anti-CSF1R (1:500, sc-692, Santa Cruz Biotechnology, USA).

Techniques: Software, Expressing, Immunofluorescence, Marker, Injection

Journal: Cell reports

Article Title: Microglia are Indispensable for Synaptic Plasticity in the Spinal Dorsal Horn and Chronic Pain

doi: 10.1016/j.celrep.2019.05.087

Figure Lengend Snippet: (A) HFS upregulated BDNF and CGRP levels in SDH, and the effects were prevented by intrathecal injection of anti-CSF1 (40.0 ng/μl) 30 min before HFS (n = 3–6 mice/group). ***P < 0.001 vs. control sham group. ##P < 0.01; ###P < 0.001 vs. control HFS group; $P < 0.05 vs. anti-CSF1 sham group, one-way ANOVA with Fisher’s LSD test. (B-D) In cultured spinal cord slices, CSF1 (1 μg/ml) markedly upregulated microglial markers (CD11b and Iba1) and CSF1R, and the effects were dose-dependently suppressed by p38 MAPK inhibitor SB203580 (10 μM) applied 1 hour prior to CSF1. Scale bar: 100 μm. n = 4–5 samples/group,* P < 0.05, ***P < 0.001 vs control group; ###P < 0.001 vs CSF1 group, one-way ANOVA with Tukey’s post-hoc test. (E-F) In culture spinal cord slices, treatment with CSF1 for 6 hours upregulated CSF1R, the phospho-p38 MAPK (p-p38) and BDNF, as well as increased BDNF in culture media. Pretreatment with p38 inhibitor SB203580 (10 μM) abolished the effects of CSF1. n = 3–5 samples/group, **P < 0.01, ***P < 0.001 vs control group; ##P < 0.01 vs CSF1 group, one-way ANOVA with Tukey’s post-hoc test. (G) Intrathecal CSF1 upregulated CGRP expression in BDNF+/+ mice but not in mice deficient of microglial BDNF (BDNF−/−), as measured 3 days after injection (n = 3–6 mice/group). ***P < 0.001 vs. BDNF+/+PBS group, ###P < 0.001 vs. BDNF+/+ CSF1 group, one-way ANOVA with Fisher’s LSD test. (H) The mechanical allodynia induced by intrathecal CSF1 was significantly attenuated in microglial BDNF deletion mice compared with control mice (n = 5–6 mice/group, ###P < 0.001 vs. BDNF+/+ CSF1 group), two-way ANOVA with Fisher’s LSD test.

Article Snippet: The tissue sections/cultured DRG neurons were first blocked with 5% donkey serum in 0.3% Triton X-100 (Sigma) for 60/30 min at room temperature (RT), and then incubated overnight at 4°C with a mixture of rabbit anti-ATF3 (1:500, sc-188, Santa Cruz Biotechnology, USA), mouse anti-CGRP antibody [4901] (1:2000, ab81887, Abcam, USA), isolectin B4-FITC (1:100, L2895, Sigma, USA), rabbit anti-GAP43 antibody (1:500, ab128005, Abcam, USA), rabbit anti-Substance P receptor antibody (1:500, AB5060, Millipore, USA.), rabbit anti-Iba1 (1:2000, 019–19741, Wako), goat anti-M-CSF antibody (1:500, AF416, R&D Systems, USA); rat anti-CD11b (1:500, 101202, Biolegend, USA); goat anti-Iba1 (1:500, ab5076, Abcam, USA) or rabbit anti-CSF1R (1:500, sc-692, Santa Cruz Biotechnology, USA).

Techniques: Injection, Cell Culture, Expressing

Journal: Cell reports

Article Title: Microglia are Indispensable for Synaptic Plasticity in the Spinal Dorsal Horn and Chronic Pain

doi: 10.1016/j.celrep.2019.05.087

Figure Lengend Snippet: (A) Western blots show the time course of Iba1 expression level after 10V HFS (n = 3 mice/ group). ***P< 0.001 vs. sham group, one-way ANOVA with Fisher’s LSD test. (B, C) Representative images of Iba1 immunostaining and the histogram show that the number of Iba1+ microglia were increased in the ipsilateral SDH at 1, 3 and 7 days after 10V HFS, and 7 days after SNI. Iba1+ cells were increased only in ipsilateral but not in contralateral SDH 7 days after HFS (7dI and 7dC). Scale bar: 50 μm. n = 3 mice/group, 2–3 sections/mouse. ***P < 0.001, ****P < 0.0001vs. sham group, ###P < 0.001 vs. HFS 7d group, one-way ANOVA with Fisher’s LSD test. (D) Experiment designs for control or microglial ablation (MG abl) at 3 or 21 days after vehicle (vehi, corn oil) diphtheria toxin treatment in CX3CR1CreER/+: R26iDTR/+mice. DT: diphtheria toxin, TM: Tamoxifen, Pain Beha: pain behavior test, EP: electrophysiology. In DT 3d HFS or DT 21d HFS group, DT was injected 3 weeks after TM and HFS was delivered at 3 or 21 days after last application of DT. In control group, vehicle (Vehi) but not DT was injected following TM injection. (E) The immunofluorescent staining showed that microglia were largely depleted at 3 days after DT treatment (DT 3d), and then fully repopulated within 21 days after DT treatment (DT 21d) in CX3CR1CreER/+: R26iDTR/+ mice. Boxed areas are magnified in the right panel. Scale bars: 100 μm (left) and 25 μm (right). (F) HFS was able to induce LTP at 21 days but not at 3 days after DT injection (n = 12 mice for DT 3 d group, n = 6–7 mice for other groups, two-way ANOVA with Tukey’s test). Representative traces of evoked C-fiber fEPSP are shown above before (i, grey line) and 3 h after 10V HFS (ii, colored line). Scale bars: x = 100 ms, y = 0.15 mV. The arrow indicates the time point at which HFS was delivered. (G) The input-output curves of fEPSP measured 7 d after HFS in mice treated with DT (for microglial ablation) and with vehicles are shown (n = 5 mice for sham group, n = 5 or 7 mice for MG ablation groups). (H, I) The increase in CGRP+ terminals induced by HFS was substantially attenuated in microglia ablation mice (n = 3 mice/group, 2–3 sections/mouse). Scale bar: 20 μm. ***P < 0.001 vs. control sham group; #P < 0.05, ##P < 0.01 vs. control HFS 3d or 7d group, one-way ANOVA with Fisher’s LSD test. (J) HFS induced mechanical allodynia in mice with microglia full repopulation (DT 21d), but not in mice with microglia ablation (DT 3d). n = 12 mice in MG ablation DT 3d group, n = 8 mice for other groups. **P < 0.01, ***P < 0.001 vs. Control HFS, two-way ANOVA with Fisher’s LSD test.

Article Snippet: The tissue sections/cultured DRG neurons were first blocked with 5% donkey serum in 0.3% Triton X-100 (Sigma) for 60/30 min at room temperature (RT), and then incubated overnight at 4°C with a mixture of rabbit anti-ATF3 (1:500, sc-188, Santa Cruz Biotechnology, USA), mouse anti-CGRP antibody [4901] (1:2000, ab81887, Abcam, USA), isolectin B4-FITC (1:100, L2895, Sigma, USA), rabbit anti-GAP43 antibody (1:500, ab128005, Abcam, USA), rabbit anti-Substance P receptor antibody (1:500, AB5060, Millipore, USA.), rabbit anti-Iba1 (1:2000, 019–19741, Wako), goat anti-M-CSF antibody (1:500, AF416, R&D Systems, USA); rat anti-CD11b (1:500, 101202, Biolegend, USA); goat anti-Iba1 (1:500, ab5076, Abcam, USA) or rabbit anti-CSF1R (1:500, sc-692, Santa Cruz Biotechnology, USA).

Techniques: Western Blot, Expressing, Immunostaining, Injection, Staining

Journal: Cell reports

Article Title: Microglia are Indispensable for Synaptic Plasticity in the Spinal Dorsal Horn and Chronic Pain

doi: 10.1016/j.celrep.2019.05.087

Figure Lengend Snippet: (A, B) The representative images and statistical analysis show the expression of CSF1 in the ipsilateral L4 DRG from 1 to 7 days after HFS and 7 days after SNI. The triple staining images showed the colocalization of CSF1, CGRP and ATF3. The arrowheads indicate that the injured DRG neurons (ATF3+) that strongly express CSF1 does not express CGRP. The arrows indicate the uninjured neurons (ATF3−) that weakly express CSF1 express CGRP following HFS or SNI. Scale bar: 50 μm. n = 3 mice/group, n = 2–3 images/mouse, *P < 0.05, ***P < 0.001 vs sham group; ###P < 0.001, compared with HFS 7d group, one-way ANOVA with Tukey’s post-hoc test. (C) Triple staining show CSF1 in SDH was co-localized with CGRP but not with IB4 at 3 days after HFS. Scale bar: 20 μm. (D) qRT-PCR analysis for CSF1 mRNA in the ipsilateral L4–5 DRGs from different groups (n = 7–12 mice/group). Values are presented as means ± SEM. *P < 0.05, **P < 0.01, ****P < 0.00001 vs. sham group; ###P < 0.001, compared with HFS 7d group, one-way ANOVA with Fisher’s LSD test. (E) Western blot and statistical analysis show that treatment of cultured DRG neurons with 40 mM KCl for 24 h increased CSF1 in culture media. n = 6 samples/group, **P < 0.01 vs Vehi group, unpaired Student’s t-test. (F, G) Immunostaining shows CSF1R in microglia was upregulated in the ipsilateral dorsal horn at 1 and 3 days after HFS (n = 3 mice/group, 2–3 sections/mouse). Scale bar: 50 μm. ***P < 0.001, compared with sham group, ###P < 0.001, compared with HFS 7d, one-way ANOVA with Tukey’s post-hoc test. (H) Western blots illustrate the increased expression of CSF1R and Iba1 protein levels at 3 days after 10V HFS, and the changes were blocked by intrathecal injection of CSF1 neutralizing antibody (anti-CSF1, 40 ng/μl, 5 μl) 30 min before HFS (n = 3–4 mice/group). **P < 0.01, ***P < 0.001 vs. sham group. ###P < 0.001 vs. control HFS group, one-way ANOVA with Fisher’s LSD test. (I) Local application of anti-CSF1 (40 ng/μl, 20 μl) onto the dorsal surface of spinal cord at recording segments 30 min before HFS did not affect baseline of C-fiber responses, but blocked late-phase of the spinal LTP (n = 5–6 mice/group). Original traces of C-fiber fEPSPs from different groups, recorded at the baseline (i), 30 min before (ii) and 3 h (iii) after HFS, are shown on the top. Scale bars: x = 100 ms, y = 0.2 mV. The arrow indicates the time point at which conditional stimulation was delivered. (J) Intrathecal injection of anti-CSF1 antibody 30 min before HFS prevented the development of mechanical hypersensitivity (n = 6 mice/group, ***P < 0.001 vs. vehicle group (Vehi), two-way ANOVA with Fisher’s LSD test.

Article Snippet: The tissue sections/cultured DRG neurons were first blocked with 5% donkey serum in 0.3% Triton X-100 (Sigma) for 60/30 min at room temperature (RT), and then incubated overnight at 4°C with a mixture of rabbit anti-ATF3 (1:500, sc-188, Santa Cruz Biotechnology, USA), mouse anti-CGRP antibody [4901] (1:2000, ab81887, Abcam, USA), isolectin B4-FITC (1:100, L2895, Sigma, USA), rabbit anti-GAP43 antibody (1:500, ab128005, Abcam, USA), rabbit anti-Substance P receptor antibody (1:500, AB5060, Millipore, USA.), rabbit anti-Iba1 (1:2000, 019–19741, Wako), goat anti-M-CSF antibody (1:500, AF416, R&D Systems, USA); rat anti-CD11b (1:500, 101202, Biolegend, USA); goat anti-Iba1 (1:500, ab5076, Abcam, USA) or rabbit anti-CSF1R (1:500, sc-692, Santa Cruz Biotechnology, USA).

Techniques: Expressing, Staining, Quantitative RT-PCR, Western Blot, Cell Culture, Immunostaining, Injection

Journal: Cell reports

Article Title: Microglia are Indispensable for Synaptic Plasticity in the Spinal Dorsal Horn and Chronic Pain

doi: 10.1016/j.celrep.2019.05.087

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The tissue sections/cultured DRG neurons were first blocked with 5% donkey serum in 0.3% Triton X-100 (Sigma) for 60/30 min at room temperature (RT), and then incubated overnight at 4°C with a mixture of rabbit anti-ATF3 (1:500, sc-188, Santa Cruz Biotechnology, USA), mouse anti-CGRP antibody [4901] (1:2000, ab81887, Abcam, USA), isolectin B4-FITC (1:100, L2895, Sigma, USA), rabbit anti-GAP43 antibody (1:500, ab128005, Abcam, USA), rabbit anti-Substance P receptor antibody (1:500, AB5060, Millipore, USA.), rabbit anti-Iba1 (1:2000, 019–19741, Wako), goat anti-M-CSF antibody (1:500, AF416, R&D Systems, USA); rat anti-CD11b (1:500, 101202, Biolegend, USA); goat anti-Iba1 (1:500, ab5076, Abcam, USA) or rabbit anti-CSF1R (1:500, sc-692, Santa Cruz Biotechnology, USA).

Techniques: Recombinant, Software, Microscopy