Structured Review

Promega ß tubulin iii
Differentiation assays. (a) Left: Adipocyte with lipid vacuole resulted from adipogenic differentiation of CD146 positive cell stained with oil red. Right: Expression of PAPR‐γ2 and aP‐2 is shown following adipogenic differentiation using PCR. (b) Left: Mineralization and appropriate morphological changes are shown following osteogenic differentiation stained with alizarin red. Right: With osteogenic differentiation, expression of OPN and Col1α1 is revealed by PCR. (c) Left: With neurogenic differentiation typical dendritic cells which express appeared Right: <t>ß‐tubulin</t> III revealed by immune‐fluorescent staining. (d) Left: With hepatocytic differentiation, polygonal/flattened shape cells appeared at day 21 (differentiation step 2) Right: Hepatogenic differentiation was confirmed by qRT‐PCR as hepatogenic related genes were upregulated postdifferentiation, specially ALB and HNF with approximately 10‐ and 2.5‐fold higher expression after differentiation. The bars represent gene expressions before and after differentiation
ß Tubulin Iii, supplied by Promega, used in various techniques. Bioz Stars score: 97/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Differential expression of drug resistance genes in CD146 positive dental pulp derived stem cells and CD146 negative fibroblasts, et al. Differential expression of drug resistance genes in CD146 positive dental pulp derived stem cells and CD146 negative fibroblasts"

Article Title: Differential expression of drug resistance genes in CD146 positive dental pulp derived stem cells and CD146 negative fibroblasts, et al. Differential expression of drug resistance genes in CD146 positive dental pulp derived stem cells and CD146 negative fibroblasts

Journal: Clinical and Experimental Dental Research

doi: 10.1002/cre2.297

Differentiation assays. (a) Left: Adipocyte with lipid vacuole resulted from adipogenic differentiation of CD146 positive cell stained with oil red. Right: Expression of PAPR‐γ2 and aP‐2 is shown following adipogenic differentiation using PCR. (b) Left: Mineralization and appropriate morphological changes are shown following osteogenic differentiation stained with alizarin red. Right: With osteogenic differentiation, expression of OPN and Col1α1 is revealed by PCR. (c) Left: With neurogenic differentiation typical dendritic cells which express appeared Right: ß‐tubulin III revealed by immune‐fluorescent staining. (d) Left: With hepatocytic differentiation, polygonal/flattened shape cells appeared at day 21 (differentiation step 2) Right: Hepatogenic differentiation was confirmed by qRT‐PCR as hepatogenic related genes were upregulated postdifferentiation, specially ALB and HNF with approximately 10‐ and 2.5‐fold higher expression after differentiation. The bars represent gene expressions before and after differentiation
Figure Legend Snippet: Differentiation assays. (a) Left: Adipocyte with lipid vacuole resulted from adipogenic differentiation of CD146 positive cell stained with oil red. Right: Expression of PAPR‐γ2 and aP‐2 is shown following adipogenic differentiation using PCR. (b) Left: Mineralization and appropriate morphological changes are shown following osteogenic differentiation stained with alizarin red. Right: With osteogenic differentiation, expression of OPN and Col1α1 is revealed by PCR. (c) Left: With neurogenic differentiation typical dendritic cells which express appeared Right: ß‐tubulin III revealed by immune‐fluorescent staining. (d) Left: With hepatocytic differentiation, polygonal/flattened shape cells appeared at day 21 (differentiation step 2) Right: Hepatogenic differentiation was confirmed by qRT‐PCR as hepatogenic related genes were upregulated postdifferentiation, specially ALB and HNF with approximately 10‐ and 2.5‐fold higher expression after differentiation. The bars represent gene expressions before and after differentiation

Techniques Used: Staining, Expressing, Polymerase Chain Reaction, Quantitative RT-PCR

2) Product Images from "Mild and repetitive very mild axonal stretch injury triggers cystoskeletal mislocalization and growth cone collapse"

Article Title: Mild and repetitive very mild axonal stretch injury triggers cystoskeletal mislocalization and growth cone collapse

Journal: PLoS ONE

doi: 10.1371/journal.pone.0176997

Immunocytochemistry images of growth cones of control (uninjured), 0.5% stretched and 5% stretched axons at 10 DIV and 72 h PI. (A-C) In the control, growth cones with distinct filopodia were apparent (arrow). The β III tubulin labelling (green) was distributed predominantly within the central domain of the growth cone while F actin (red) was confined to the peripheral region. (D-F) In the 0.5% stretched axon, microtubules appeared to form a loop in the central region of the growth cones and F-actin was confined to the peripheral and transition region only. (G-I) In the 5% stretched axon, F-actin was most abundant in the axon tip, forming bulb like accumulations. Scale Bars = 10 μ m.
Figure Legend Snippet: Immunocytochemistry images of growth cones of control (uninjured), 0.5% stretched and 5% stretched axons at 10 DIV and 72 h PI. (A-C) In the control, growth cones with distinct filopodia were apparent (arrow). The β III tubulin labelling (green) was distributed predominantly within the central domain of the growth cone while F actin (red) was confined to the peripheral region. (D-F) In the 0.5% stretched axon, microtubules appeared to form a loop in the central region of the growth cones and F-actin was confined to the peripheral and transition region only. (G-I) In the 5% stretched axon, F-actin was most abundant in the axon tip, forming bulb like accumulations. Scale Bars = 10 μ m.

Techniques Used: Immunocytochemistry

Double immunolabelling verified extensive network of axons ( β III tubulin immunoreactivity) and growth cones (F-actin staining) within the axon compartment of the stretch injury model at 7 DIV. (A) Axons (green; β III tubulin) of primary rat cortical neurons extended into the axon compartment through microgrooves (450 μ m long, 10 μ m width and 3 μ m high) at 7 DIV. Dashed lines indicate the location of the pneumatic channel (or stretch injury) and solid lines show the microgrooves region. (B) High magnification of axons with growth cones immunostained with F-actin (red) and microtubules (green) (white square box in panel A). Insets show higher magnification of growth cone. Scale Bars = 200 μ m (A), 75 μ m (B), 40 μ m (insets).
Figure Legend Snippet: Double immunolabelling verified extensive network of axons ( β III tubulin immunoreactivity) and growth cones (F-actin staining) within the axon compartment of the stretch injury model at 7 DIV. (A) Axons (green; β III tubulin) of primary rat cortical neurons extended into the axon compartment through microgrooves (450 μ m long, 10 μ m width and 3 μ m high) at 7 DIV. Dashed lines indicate the location of the pneumatic channel (or stretch injury) and solid lines show the microgrooves region. (B) High magnification of axons with growth cones immunostained with F-actin (red) and microtubules (green) (white square box in panel A). Insets show higher magnification of growth cone. Scale Bars = 200 μ m (A), 75 μ m (B), 40 μ m (insets).

Techniques Used: Staining

Graphs showing the mean percentages of collapse growth cone and the extent of colocalizatuon of F actin and β III tubulin in growth cones of control cultures and cultures after 0.5% stretched, 5% stretched and repetitive very mild (2×0.5%) stretched axons at different time point. (A) Stretch injury induced increased axonal growth cone collapsed at both 24 h and 72 h PI compared to the control. In addition, repetitive very mild (2×0.5%) stretch injury induced more collapsed growth cones when compared to single 0.5% stretched axon at 72 h PI. (B) The growth cones in 5% stretched axon had significantly higher colocalization value of β III tubulin and F-actin compared to both the growth cones in control and 0.5% stretched axon at 72 h PI. However, there was no significant difference between the growth cones in control, 0.5% stretched or 5% stretched axon at 24 h PI. The growth cones in 2×0.5% repetitive stretched axon has significantly higher colocalization value of β III tubulin and F-actin if compare to both the growth cones in control and single 0.5% stretched axon at 72 h PI. *p
Figure Legend Snippet: Graphs showing the mean percentages of collapse growth cone and the extent of colocalizatuon of F actin and β III tubulin in growth cones of control cultures and cultures after 0.5% stretched, 5% stretched and repetitive very mild (2×0.5%) stretched axons at different time point. (A) Stretch injury induced increased axonal growth cone collapsed at both 24 h and 72 h PI compared to the control. In addition, repetitive very mild (2×0.5%) stretch injury induced more collapsed growth cones when compared to single 0.5% stretched axon at 72 h PI. (B) The growth cones in 5% stretched axon had significantly higher colocalization value of β III tubulin and F-actin compared to both the growth cones in control and 0.5% stretched axon at 72 h PI. However, there was no significant difference between the growth cones in control, 0.5% stretched or 5% stretched axon at 24 h PI. The growth cones in 2×0.5% repetitive stretched axon has significantly higher colocalization value of β III tubulin and F-actin if compare to both the growth cones in control and single 0.5% stretched axon at 72 h PI. *p

Techniques Used:

3) Product Images from "EVIDENCE FOR DYING-BACK AXONAL DEGENERATION IN AGE-ASSOCIATED SKELETAL MUSCLE DECLINE"

Article Title: EVIDENCE FOR DYING-BACK AXONAL DEGENERATION IN AGE-ASSOCIATED SKELETAL MUSCLE DECLINE

Journal: Muscle & nerve

doi: 10.1002/mus.25267

Partial denervation of NMJs in soleus muscles of young and old mice. Double immunofluorescence staining was performed (green = β III-tubulin; red =bungarotoxin), and the confocal images were analyzed. (A) NMJ of soleus muscle in a young mouse. Note that postsynaptic motor endplates (yellow arrow) appear well-organized, distinct, and compact, with presynaptic axons covering wide areas of motor endplates. (B) NMJ of soleus muscle in an old mouse. Postsynaptic motor endplates are fragmented and disorganized (yellow arrow). Diameters of the motor endplates are larger, and presynaptic axons do not cover the entire areas of the motor end-plates, suggesting partial denervation (yellow arrow). (C) The percentage coverage of presynaptic axons over the postsynaptic motor endplate (AChR occupancy) was calculated using confocal imaging software; the results show significantly greater coverage in the young mice ( N =5, 91.93%) than in the old mice ( N =5, 40.97%). (D) Comparison of postsynaptic endplate area. The postsynaptic endplate areas are much greater in the old mice ( N =5, average 497.74 μ m 2 than in the young mice ( N =5, average 299.76 μ m 2 . Representative images of completely denervated NMJs in old mice are seen in panels (E) and (F) (green = β III-tubulin; red =bungarotoxin).
Figure Legend Snippet: Partial denervation of NMJs in soleus muscles of young and old mice. Double immunofluorescence staining was performed (green = β III-tubulin; red =bungarotoxin), and the confocal images were analyzed. (A) NMJ of soleus muscle in a young mouse. Note that postsynaptic motor endplates (yellow arrow) appear well-organized, distinct, and compact, with presynaptic axons covering wide areas of motor endplates. (B) NMJ of soleus muscle in an old mouse. Postsynaptic motor endplates are fragmented and disorganized (yellow arrow). Diameters of the motor endplates are larger, and presynaptic axons do not cover the entire areas of the motor end-plates, suggesting partial denervation (yellow arrow). (C) The percentage coverage of presynaptic axons over the postsynaptic motor endplate (AChR occupancy) was calculated using confocal imaging software; the results show significantly greater coverage in the young mice ( N =5, 91.93%) than in the old mice ( N =5, 40.97%). (D) Comparison of postsynaptic endplate area. The postsynaptic endplate areas are much greater in the old mice ( N =5, average 497.74 μ m 2 than in the young mice ( N =5, average 299.76 μ m 2 . Representative images of completely denervated NMJs in old mice are seen in panels (E) and (F) (green = β III-tubulin; red =bungarotoxin).

Techniques Used: Mouse Assay, Double Immunofluorescence Staining, Imaging, Software

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    Promega ß tubulin iii
    Differentiation assays. (a) Left: Adipocyte with lipid vacuole resulted from adipogenic differentiation of CD146 positive cell stained with oil red. Right: Expression of PAPR‐γ2 and aP‐2 is shown following adipogenic differentiation using PCR. (b) Left: Mineralization and appropriate morphological changes are shown following osteogenic differentiation stained with alizarin red. Right: With osteogenic differentiation, expression of OPN and Col1α1 is revealed by PCR. (c) Left: With neurogenic differentiation typical dendritic cells which express appeared Right: <t>ß‐tubulin</t> III revealed by immune‐fluorescent staining. (d) Left: With hepatocytic differentiation, polygonal/flattened shape cells appeared at day 21 (differentiation step 2) Right: Hepatogenic differentiation was confirmed by qRT‐PCR as hepatogenic related genes were upregulated postdifferentiation, specially ALB and HNF with approximately 10‐ and 2.5‐fold higher expression after differentiation. The bars represent gene expressions before and after differentiation
    ß Tubulin Iii, supplied by Promega, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega β iii tubulin
    Localization of C9ORF72 over development in vitro . Immunofluorescence was carried out on primary cultured cortical neurons. a At 1 DIV, C9ORF72 ( red ) labeling was present within cell bodies, excluding nuclei (DAPI, blue ) and punctate localization was present in neurites and growth cones <t>(β-III</t> <t>tubulin,</t> green ). b Co-staining with phalloidin ( green ) at 3 DIV confirmed localization of C9ORF72 ( red ) labeling to growth cones and to filopodia ( arrows ). c At 14 DIV, C9ORF72 ( red ) was localized to nuclei of a population of neurons ( arrows ) but was less intensely expressed in nuclei of other neurons ( arrowhead ). Neurons indicated by MAP2 ( green ). Neurons with nuclear immunolabeling for C9ORF72 frequently had punctate somal localization of this protein. Inset ( c ) shows C9ORF72 labeling in nuclei and in puncta in surrounding cytoplasm. Scale bar: A, 2.5 μm; B, C, 10 μm
    β Iii Tubulin, supplied by Promega, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega mouse anti beta iii tubulin
    PTX3 does not affect synapse number and organization Representative images of 14DIV control and PTX3‐treated cultures stained for the presynaptic marker Bassoon (blue), the postsynaptic protein PSD95 (green), and the microtubule protein <t>tubulin</t> (red). Scale bar: 5 μm. Quantification of synaptic density shows no differences either for postsynaptic marker (PSD95/μm), or for presynaptic marker (Bsn/μm) or as a total number of synapses (PSD95 Bsn/μm) in control and PTX3‐treated cultures (PSD95/μm, Ctr = 0.391 ± 0.018; PTX3 = 0.379 ± 0.016; Bsn/μm, Ctr = 0.291 ± 0.012, PTX3 = 0.311 ± 0.012; PSD95 Bsn/μm, Ctr = 0.218 ± 0.009, PTX3 = 0.235 ± 0.013. Number of dendrites: 109 Ctr, 107 PTX3, Mann–Whitney test; five independent experiments, data are presented as mean ± SEM). Quantitative analysis of the mean size of PSD95 and Bsn puncta shows no differences in control or PTX3‐treated neurons (in μm 2 , PSD95: Ctr = 0.155 ± 0.010; PTX3 = 0.158 ± 0.008; Bsn: Ctr = 0.205 ± 0.013, PTX3 = 0.214 ± 0.012; Number of dendrites: 134 ctr, 124 PTX3, Mann–Whitney test; five independent experiments, data are presented as mean ± SEM). (D) Representative images of GFP‐expressing dendritic branches of control and PTX3‐treated neurons and (E) quantification of dendritic spine density, i.e., number of spines per μm of parent dendrite (Ctr = 0.607 ± 0.020, PTX3 = 0.598 ± 0.021, number of examined dendrites: 73 and 71 respectively; Mann–Whitney test; <t>three</t> independent experiments, data are presented as mean ± SEM). Scale bar: 5 μm. Western blotting analysis of major synaptic proteins on lysates from control or PTX3‐treated cultures. GAPDH was used as reference marker. Chronic PTX3 administration does not increase neither synaptic density nor synaptic puncta size. (G) Representative images of 14DIV control and PTX3‐treated cultures stained for the presynaptic marker Bassoon (blue), and the postsynaptic protein PSD95 (green) and tubulin (red). Scale bar: 5 μm. (H) Quantification of synaptic density parameters (PSD95/μm; Bsn/μm; PSD95 Bsn/μm) in control and PTX3‐treated neurons (PSD95/μm, Ctr = 0.297 ± 0.029; PTX3 = 0.334 ± 0.026; Bsn/μm, Ctr = 0.261 ± 0.021, PTX3 = 0.221 ± 0.020; PSD95 Bsn/μm, Ctr = 0.209 ± 0.019, PTX3 = 0.148 ± 0.014. Number of dendrites: Ctr = 20, PTX3 = 40, Mann–Whitney test; three independent experiments; data represented as mean ± SEM). (I) Analysis of the mean size of PSD95 and Bsn puncta shows no differences in control or PTX3‐treated neurons (in μm 2 , PSD95: Ctr = 0.081 ± 0.009; PTX3 = 0.094 ± 0.010; Bsn: Ctr = 0.175 ± 0.021, PTX3 = 0.138 ± 0.016; number of dendrites: Ctr = 22, PTX3 = 48, Mann–Whitney test; three independent experiments, data represented as mean ± SEM). Source data are available online for this figure.
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    Differentiation assays. (a) Left: Adipocyte with lipid vacuole resulted from adipogenic differentiation of CD146 positive cell stained with oil red. Right: Expression of PAPR‐γ2 and aP‐2 is shown following adipogenic differentiation using PCR. (b) Left: Mineralization and appropriate morphological changes are shown following osteogenic differentiation stained with alizarin red. Right: With osteogenic differentiation, expression of OPN and Col1α1 is revealed by PCR. (c) Left: With neurogenic differentiation typical dendritic cells which express appeared Right: ß‐tubulin III revealed by immune‐fluorescent staining. (d) Left: With hepatocytic differentiation, polygonal/flattened shape cells appeared at day 21 (differentiation step 2) Right: Hepatogenic differentiation was confirmed by qRT‐PCR as hepatogenic related genes were upregulated postdifferentiation, specially ALB and HNF with approximately 10‐ and 2.5‐fold higher expression after differentiation. The bars represent gene expressions before and after differentiation

    Journal: Clinical and Experimental Dental Research

    Article Title: Differential expression of drug resistance genes in CD146 positive dental pulp derived stem cells and CD146 negative fibroblasts, et al. Differential expression of drug resistance genes in CD146 positive dental pulp derived stem cells and CD146 negative fibroblasts

    doi: 10.1002/cre2.297

    Figure Lengend Snippet: Differentiation assays. (a) Left: Adipocyte with lipid vacuole resulted from adipogenic differentiation of CD146 positive cell stained with oil red. Right: Expression of PAPR‐γ2 and aP‐2 is shown following adipogenic differentiation using PCR. (b) Left: Mineralization and appropriate morphological changes are shown following osteogenic differentiation stained with alizarin red. Right: With osteogenic differentiation, expression of OPN and Col1α1 is revealed by PCR. (c) Left: With neurogenic differentiation typical dendritic cells which express appeared Right: ß‐tubulin III revealed by immune‐fluorescent staining. (d) Left: With hepatocytic differentiation, polygonal/flattened shape cells appeared at day 21 (differentiation step 2) Right: Hepatogenic differentiation was confirmed by qRT‐PCR as hepatogenic related genes were upregulated postdifferentiation, specially ALB and HNF with approximately 10‐ and 2.5‐fold higher expression after differentiation. The bars represent gene expressions before and after differentiation

    Article Snippet: To confirm neural differentiation, immunostaining was performed for ß‐tubulin III (Promega cat number: G7121) as a specific antibody against neurons.

    Techniques: Staining, Expressing, Polymerase Chain Reaction, Quantitative RT-PCR

    Localization of C9ORF72 over development in vitro . Immunofluorescence was carried out on primary cultured cortical neurons. a At 1 DIV, C9ORF72 ( red ) labeling was present within cell bodies, excluding nuclei (DAPI, blue ) and punctate localization was present in neurites and growth cones (β-III tubulin, green ). b Co-staining with phalloidin ( green ) at 3 DIV confirmed localization of C9ORF72 ( red ) labeling to growth cones and to filopodia ( arrows ). c At 14 DIV, C9ORF72 ( red ) was localized to nuclei of a population of neurons ( arrows ) but was less intensely expressed in nuclei of other neurons ( arrowhead ). Neurons indicated by MAP2 ( green ). Neurons with nuclear immunolabeling for C9ORF72 frequently had punctate somal localization of this protein. Inset ( c ) shows C9ORF72 labeling in nuclei and in puncta in surrounding cytoplasm. Scale bar: A, 2.5 μm; B, C, 10 μm

    Journal: Acta Neuropathologica Communications

    Article Title: C9ORF72 expression and cellular localization over mouse development

    doi: 10.1186/s40478-015-0238-7

    Figure Lengend Snippet: Localization of C9ORF72 over development in vitro . Immunofluorescence was carried out on primary cultured cortical neurons. a At 1 DIV, C9ORF72 ( red ) labeling was present within cell bodies, excluding nuclei (DAPI, blue ) and punctate localization was present in neurites and growth cones (β-III tubulin, green ). b Co-staining with phalloidin ( green ) at 3 DIV confirmed localization of C9ORF72 ( red ) labeling to growth cones and to filopodia ( arrows ). c At 14 DIV, C9ORF72 ( red ) was localized to nuclei of a population of neurons ( arrows ) but was less intensely expressed in nuclei of other neurons ( arrowhead ). Neurons indicated by MAP2 ( green ). Neurons with nuclear immunolabeling for C9ORF72 frequently had punctate somal localization of this protein. Inset ( c ) shows C9ORF72 labeling in nuclei and in puncta in surrounding cytoplasm. Scale bar: A, 2.5 μm; B, C, 10 μm

    Article Snippet: Immunofluorescence labeling was carried out for both cultured cells and brain tissue following standard procedures using antibodies against C9ORF72 (as above), β-III Tubulin (1:5000, Promega) and MAP2 (1:1000, Millipore) diluted in PBS with 0.6 % Triton-X-100 and incubated at RT overnight.

    Techniques: In Vitro, Immunofluorescence, Cell Culture, Labeling, Staining, Immunolabeling

    PTX3 does not affect synapse number and organization Representative images of 14DIV control and PTX3‐treated cultures stained for the presynaptic marker Bassoon (blue), the postsynaptic protein PSD95 (green), and the microtubule protein tubulin (red). Scale bar: 5 μm. Quantification of synaptic density shows no differences either for postsynaptic marker (PSD95/μm), or for presynaptic marker (Bsn/μm) or as a total number of synapses (PSD95 Bsn/μm) in control and PTX3‐treated cultures (PSD95/μm, Ctr = 0.391 ± 0.018; PTX3 = 0.379 ± 0.016; Bsn/μm, Ctr = 0.291 ± 0.012, PTX3 = 0.311 ± 0.012; PSD95 Bsn/μm, Ctr = 0.218 ± 0.009, PTX3 = 0.235 ± 0.013. Number of dendrites: 109 Ctr, 107 PTX3, Mann–Whitney test; five independent experiments, data are presented as mean ± SEM). Quantitative analysis of the mean size of PSD95 and Bsn puncta shows no differences in control or PTX3‐treated neurons (in μm 2 , PSD95: Ctr = 0.155 ± 0.010; PTX3 = 0.158 ± 0.008; Bsn: Ctr = 0.205 ± 0.013, PTX3 = 0.214 ± 0.012; Number of dendrites: 134 ctr, 124 PTX3, Mann–Whitney test; five independent experiments, data are presented as mean ± SEM). (D) Representative images of GFP‐expressing dendritic branches of control and PTX3‐treated neurons and (E) quantification of dendritic spine density, i.e., number of spines per μm of parent dendrite (Ctr = 0.607 ± 0.020, PTX3 = 0.598 ± 0.021, number of examined dendrites: 73 and 71 respectively; Mann–Whitney test; three independent experiments, data are presented as mean ± SEM). Scale bar: 5 μm. Western blotting analysis of major synaptic proteins on lysates from control or PTX3‐treated cultures. GAPDH was used as reference marker. Chronic PTX3 administration does not increase neither synaptic density nor synaptic puncta size. (G) Representative images of 14DIV control and PTX3‐treated cultures stained for the presynaptic marker Bassoon (blue), and the postsynaptic protein PSD95 (green) and tubulin (red). Scale bar: 5 μm. (H) Quantification of synaptic density parameters (PSD95/μm; Bsn/μm; PSD95 Bsn/μm) in control and PTX3‐treated neurons (PSD95/μm, Ctr = 0.297 ± 0.029; PTX3 = 0.334 ± 0.026; Bsn/μm, Ctr = 0.261 ± 0.021, PTX3 = 0.221 ± 0.020; PSD95 Bsn/μm, Ctr = 0.209 ± 0.019, PTX3 = 0.148 ± 0.014. Number of dendrites: Ctr = 20, PTX3 = 40, Mann–Whitney test; three independent experiments; data represented as mean ± SEM). (I) Analysis of the mean size of PSD95 and Bsn puncta shows no differences in control or PTX3‐treated neurons (in μm 2 , PSD95: Ctr = 0.081 ± 0.009; PTX3 = 0.094 ± 0.010; Bsn: Ctr = 0.175 ± 0.021, PTX3 = 0.138 ± 0.016; number of dendrites: Ctr = 22, PTX3 = 48, Mann–Whitney test; three independent experiments, data represented as mean ± SEM). Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Pentraxin 3 regulates synaptic function by inducing AMPA receptor clustering via ECM remodeling and β1‐integrin

    doi: 10.15252/embj.201899529

    Figure Lengend Snippet: PTX3 does not affect synapse number and organization Representative images of 14DIV control and PTX3‐treated cultures stained for the presynaptic marker Bassoon (blue), the postsynaptic protein PSD95 (green), and the microtubule protein tubulin (red). Scale bar: 5 μm. Quantification of synaptic density shows no differences either for postsynaptic marker (PSD95/μm), or for presynaptic marker (Bsn/μm) or as a total number of synapses (PSD95 Bsn/μm) in control and PTX3‐treated cultures (PSD95/μm, Ctr = 0.391 ± 0.018; PTX3 = 0.379 ± 0.016; Bsn/μm, Ctr = 0.291 ± 0.012, PTX3 = 0.311 ± 0.012; PSD95 Bsn/μm, Ctr = 0.218 ± 0.009, PTX3 = 0.235 ± 0.013. Number of dendrites: 109 Ctr, 107 PTX3, Mann–Whitney test; five independent experiments, data are presented as mean ± SEM). Quantitative analysis of the mean size of PSD95 and Bsn puncta shows no differences in control or PTX3‐treated neurons (in μm 2 , PSD95: Ctr = 0.155 ± 0.010; PTX3 = 0.158 ± 0.008; Bsn: Ctr = 0.205 ± 0.013, PTX3 = 0.214 ± 0.012; Number of dendrites: 134 ctr, 124 PTX3, Mann–Whitney test; five independent experiments, data are presented as mean ± SEM). (D) Representative images of GFP‐expressing dendritic branches of control and PTX3‐treated neurons and (E) quantification of dendritic spine density, i.e., number of spines per μm of parent dendrite (Ctr = 0.607 ± 0.020, PTX3 = 0.598 ± 0.021, number of examined dendrites: 73 and 71 respectively; Mann–Whitney test; three independent experiments, data are presented as mean ± SEM). Scale bar: 5 μm. Western blotting analysis of major synaptic proteins on lysates from control or PTX3‐treated cultures. GAPDH was used as reference marker. Chronic PTX3 administration does not increase neither synaptic density nor synaptic puncta size. (G) Representative images of 14DIV control and PTX3‐treated cultures stained for the presynaptic marker Bassoon (blue), and the postsynaptic protein PSD95 (green) and tubulin (red). Scale bar: 5 μm. (H) Quantification of synaptic density parameters (PSD95/μm; Bsn/μm; PSD95 Bsn/μm) in control and PTX3‐treated neurons (PSD95/μm, Ctr = 0.297 ± 0.029; PTX3 = 0.334 ± 0.026; Bsn/μm, Ctr = 0.261 ± 0.021, PTX3 = 0.221 ± 0.020; PSD95 Bsn/μm, Ctr = 0.209 ± 0.019, PTX3 = 0.148 ± 0.014. Number of dendrites: Ctr = 20, PTX3 = 40, Mann–Whitney test; three independent experiments; data represented as mean ± SEM). (I) Analysis of the mean size of PSD95 and Bsn puncta shows no differences in control or PTX3‐treated neurons (in μm 2 , PSD95: Ctr = 0.081 ± 0.009; PTX3 = 0.094 ± 0.010; Bsn: Ctr = 0.175 ± 0.021, PTX3 = 0.138 ± 0.016; number of dendrites: Ctr = 22, PTX3 = 48, Mann–Whitney test; three independent experiments, data represented as mean ± SEM). Source data are available online for this figure.

    Article Snippet: The following antibodies were used: rabbit anti‐tubulin (1:100; T3526 Sigma‐Aldrich, Milan, Italy), guinea pig anti‐Bassoon (1:300; 141004, Synaptic Systems, Goettingen, Germany), mouse anti‐PSD95 (1:400; 75‐028, UC Davis/NIH NeuroMab Facility, CA), mouse anti‐gephyrin (1:500; 147021, Synaptic Systems, Goettingen, Germany), mouse anti‐beta III tubulin (1:400; G712A, Promega Corporation, Madison, USA), rabbit anti‐tubulin (1:80; Sigma‐Aldrich, Milan, Italy), rabbit anti‐aggrecan (1:200; AB1031, Millipore, Billerica, MA, USA), DAPI (1:5,000, Thermo Fisher).

    Techniques: Staining, Marker, MANN-WHITNEY, Expressing, Western Blot

    An intact perineural network is necessary for GluA recruitment to the postsynaptic membrane Low and high magnification images of control and PTX3‐treated neurons stained for the PNN main component, aggrecan (red), the synaptic proteins PSD95 (green), and Bsn (blue). Scale bar: 5 μm. PTX3 application induces a remodeling of the PNN in culture, as assessed by the increase mean intensity and integrated density value of the synapse‐co‐localizing aggrecan signal, whereas no difference in the total area of aggrecan is evident (integrated density: Ctr = 1 ± 0.103, PTX3 = 1.874 ± 0.197; mean intensity: Ctr = 1.000 ± 0.088, PTX3 = 1.556 ± 0.107; total area: Ctr = 1 ± 0.179, PTX3 = 0.982 ± 0.149. Number of fields examined: 26 Ctr, 22 PTX3; Mann–Whitney test; three independent experiments, data are presented as normalized mean values ± SEM). Overnight treatment with hyaluronidase destroys PNN as shown by immunofluorescence for aggrecan (red), DAPI (cyan), and βIII tubulin (green) and confocal analysis. Scale bar: 20 μm. Representative images showing 14DIV neurons stained for surface AMPARs (GluA, green), the presynaptic protein Bassoon (blue), and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5 μm. HAse treatment blocks PTX3‐induced synaptic surface AMPA receptors clustering (Ctr = 1.000 ± 0.075, PTX3 = 1.425 ± 0.088, PTX3+HAse = 0.961 ± 0.053, HAse = 1.080 ± 0.087. Number of fields examined: 37, 23, 53, 28, respectively; Kruskal–Wallis test, P = 0.0004 followed by post hoc Tukey test as indicated in figure; three independent experiments, data are presented as normalized mean value ± SEM). Representative images showing 14DIV WT and TSG6 KO neurons stained for surface AMPARs (GluA, green), the presynaptic protein Bassoon (blue), and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5 μm. Synaptic surface GluA quantitation showing no effect of PTX3 treatment in TSG6 KO cultures. On the contrary WT cultures (from littermates) display increased surface GluA Bsn/Bsn upon PTX3 treatment. A significant enhancement of surface GluA receptors was induced by TTX in both TSG6 KO and WT cultures (WT = 1 ± 0.06; WT+PTX3 = 1.389 ± 0.113; WT+TTX = 1.698 ± 0.109; Number of fields examined: 40, 39, 32 respectively; Kruskal–Wallis test P

    Journal: The EMBO Journal

    Article Title: Pentraxin 3 regulates synaptic function by inducing AMPA receptor clustering via ECM remodeling and β1‐integrin

    doi: 10.15252/embj.201899529

    Figure Lengend Snippet: An intact perineural network is necessary for GluA recruitment to the postsynaptic membrane Low and high magnification images of control and PTX3‐treated neurons stained for the PNN main component, aggrecan (red), the synaptic proteins PSD95 (green), and Bsn (blue). Scale bar: 5 μm. PTX3 application induces a remodeling of the PNN in culture, as assessed by the increase mean intensity and integrated density value of the synapse‐co‐localizing aggrecan signal, whereas no difference in the total area of aggrecan is evident (integrated density: Ctr = 1 ± 0.103, PTX3 = 1.874 ± 0.197; mean intensity: Ctr = 1.000 ± 0.088, PTX3 = 1.556 ± 0.107; total area: Ctr = 1 ± 0.179, PTX3 = 0.982 ± 0.149. Number of fields examined: 26 Ctr, 22 PTX3; Mann–Whitney test; three independent experiments, data are presented as normalized mean values ± SEM). Overnight treatment with hyaluronidase destroys PNN as shown by immunofluorescence for aggrecan (red), DAPI (cyan), and βIII tubulin (green) and confocal analysis. Scale bar: 20 μm. Representative images showing 14DIV neurons stained for surface AMPARs (GluA, green), the presynaptic protein Bassoon (blue), and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5 μm. HAse treatment blocks PTX3‐induced synaptic surface AMPA receptors clustering (Ctr = 1.000 ± 0.075, PTX3 = 1.425 ± 0.088, PTX3+HAse = 0.961 ± 0.053, HAse = 1.080 ± 0.087. Number of fields examined: 37, 23, 53, 28, respectively; Kruskal–Wallis test, P = 0.0004 followed by post hoc Tukey test as indicated in figure; three independent experiments, data are presented as normalized mean value ± SEM). Representative images showing 14DIV WT and TSG6 KO neurons stained for surface AMPARs (GluA, green), the presynaptic protein Bassoon (blue), and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Scale bar: 5 μm. Synaptic surface GluA quantitation showing no effect of PTX3 treatment in TSG6 KO cultures. On the contrary WT cultures (from littermates) display increased surface GluA Bsn/Bsn upon PTX3 treatment. A significant enhancement of surface GluA receptors was induced by TTX in both TSG6 KO and WT cultures (WT = 1 ± 0.06; WT+PTX3 = 1.389 ± 0.113; WT+TTX = 1.698 ± 0.109; Number of fields examined: 40, 39, 32 respectively; Kruskal–Wallis test P

    Article Snippet: The following antibodies were used: rabbit anti‐tubulin (1:100; T3526 Sigma‐Aldrich, Milan, Italy), guinea pig anti‐Bassoon (1:300; 141004, Synaptic Systems, Goettingen, Germany), mouse anti‐PSD95 (1:400; 75‐028, UC Davis/NIH NeuroMab Facility, CA), mouse anti‐gephyrin (1:500; 147021, Synaptic Systems, Goettingen, Germany), mouse anti‐beta III tubulin (1:400; G712A, Promega Corporation, Madison, USA), rabbit anti‐tubulin (1:80; Sigma‐Aldrich, Milan, Italy), rabbit anti‐aggrecan (1:200; AB1031, Millipore, Billerica, MA, USA), DAPI (1:5,000, Thermo Fisher).

    Techniques: Staining, MANN-WHITNEY, Immunofluorescence, Quantitation Assay

    PTX3 activity is inhibited by thrombospondin‐1 through direct interaction PTX3 binds TSP1 and TSP2 but not TSP4. Different amounts of human recombinant PTX3 were incubated in microplate wells coated with purified human TSP1 or recombinant TSP2 and TSP4. Binding is reported as absorbance at 450 nm (mean ± SD). Data are from one experiment out of three performed. N‐terminal PTX3 binds TSP1. Binding of PTX3 N‐term domain was performed on TSP1 immobilized on plastic wells. Data are reported as absorbance at 450 nm (mean ± SD) and are representative of one out of two experiments performed. Schematic representation of TSP1 monomer and the TSP1 proteolytic fragments (N‐term, C‐term) and recombinant domains (P123‐1, E123‐1, E123CaG‐1, Ca‐1) used in the study. PTX3 binds the C‐term proteolytic fragment of TSP1. TSP1 and its fragments C‐term, N‐term, and Ca‐1 (type III repeats) were immobilized in plastic wells (5 μg/ml) and binding of PTX3 is reported as absorbance at 450 nm (mean ± SD). Data refers to one out of two experiments performed with similar results. PTX3 and its N‐terminal domain bind TSP1 C‐terminal globular domain. 50 nM of P123‐1 (type I “properdin” repeats), E123‐1 (type II EGF repeats), E123CaG‐1 (type II repeats plus type III repeats and globular C‐terminus), and TSP1 were immobilized in plastic well. Binding with PTX3 or N‐terminal domain (both at 220 nM) was analyzed. Data are reported as absorbance at 450 nm (mean ± SD) and refer to one out of two experiments performed with similar results. Representative images of 14DIV control and PTX3‐treated cultures stained for the presynaptic marker bassoon (blue) and the microtubule protein tubulin (red). Scale bar: 10 μm. Quantification of synaptic density (Bsn/μm) in the different experimental conditions (Ctr = 0.385 ± 0.029, PTX3 = 0.407 ± 0.032, TSP1 = 0.721 ± 0.039, PTX3+TSP1 = 0.707 ± 0.037, E123 = 0.764 ± 0.047, PTX3+E123 = 0.719 ± 0.041. Number of fields examined: 86, 80, 75, 72, 46, 55 respectively; Kruskal–Wallis test, P

    Journal: The EMBO Journal

    Article Title: Pentraxin 3 regulates synaptic function by inducing AMPA receptor clustering via ECM remodeling and β1‐integrin

    doi: 10.15252/embj.201899529

    Figure Lengend Snippet: PTX3 activity is inhibited by thrombospondin‐1 through direct interaction PTX3 binds TSP1 and TSP2 but not TSP4. Different amounts of human recombinant PTX3 were incubated in microplate wells coated with purified human TSP1 or recombinant TSP2 and TSP4. Binding is reported as absorbance at 450 nm (mean ± SD). Data are from one experiment out of three performed. N‐terminal PTX3 binds TSP1. Binding of PTX3 N‐term domain was performed on TSP1 immobilized on plastic wells. Data are reported as absorbance at 450 nm (mean ± SD) and are representative of one out of two experiments performed. Schematic representation of TSP1 monomer and the TSP1 proteolytic fragments (N‐term, C‐term) and recombinant domains (P123‐1, E123‐1, E123CaG‐1, Ca‐1) used in the study. PTX3 binds the C‐term proteolytic fragment of TSP1. TSP1 and its fragments C‐term, N‐term, and Ca‐1 (type III repeats) were immobilized in plastic wells (5 μg/ml) and binding of PTX3 is reported as absorbance at 450 nm (mean ± SD). Data refers to one out of two experiments performed with similar results. PTX3 and its N‐terminal domain bind TSP1 C‐terminal globular domain. 50 nM of P123‐1 (type I “properdin” repeats), E123‐1 (type II EGF repeats), E123CaG‐1 (type II repeats plus type III repeats and globular C‐terminus), and TSP1 were immobilized in plastic well. Binding with PTX3 or N‐terminal domain (both at 220 nM) was analyzed. Data are reported as absorbance at 450 nm (mean ± SD) and refer to one out of two experiments performed with similar results. Representative images of 14DIV control and PTX3‐treated cultures stained for the presynaptic marker bassoon (blue) and the microtubule protein tubulin (red). Scale bar: 10 μm. Quantification of synaptic density (Bsn/μm) in the different experimental conditions (Ctr = 0.385 ± 0.029, PTX3 = 0.407 ± 0.032, TSP1 = 0.721 ± 0.039, PTX3+TSP1 = 0.707 ± 0.037, E123 = 0.764 ± 0.047, PTX3+E123 = 0.719 ± 0.041. Number of fields examined: 86, 80, 75, 72, 46, 55 respectively; Kruskal–Wallis test, P

    Article Snippet: The following antibodies were used: rabbit anti‐tubulin (1:100; T3526 Sigma‐Aldrich, Milan, Italy), guinea pig anti‐Bassoon (1:300; 141004, Synaptic Systems, Goettingen, Germany), mouse anti‐PSD95 (1:400; 75‐028, UC Davis/NIH NeuroMab Facility, CA), mouse anti‐gephyrin (1:500; 147021, Synaptic Systems, Goettingen, Germany), mouse anti‐beta III tubulin (1:400; G712A, Promega Corporation, Madison, USA), rabbit anti‐tubulin (1:80; Sigma‐Aldrich, Milan, Italy), rabbit anti‐aggrecan (1:200; AB1031, Millipore, Billerica, MA, USA), DAPI (1:5,000, Thermo Fisher).

    Techniques: Activity Assay, Recombinant, Incubation, Purification, Binding Assay, Staining, Marker

    PTX3 increases excitatory glutamatergic neurotransmission by promoting AMPA receptors insertion at the synapse Representative mEPSC traces recorded from control and PTX3‐treated (1 μg/ml; 48 h) neurons. mEPSC frequency quantitation (Hz, Ctr = 0.618 ± 0.069; PTX3 = 1.991 ± 0.313; number of neurons: Ctr = 22, PTX3 = 16; three independent experiments, Mann–Whitney test, data are presented as a distribution plus mean ± SEM). mEPSC amplitude quantitation and cumulative probability distribution of mEPSC amplitudes (pA, Ctr = 12.76 ± 0.813; PTX3 = 16.07 ± 0.709. Number of neurons: Ctr = 22, PTX3 = 16; three independent experiments, Mann–Whitney test. Data are presented as a distribution plus mean ± SEM). Cumulative probability distributions are analyzed by Kolmogorov–Smirnov test. Representative images showing 14DIV neurons stained for surface AMPAR (GluA, green), the presynaptic protein Bassoon (blue), and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Inset: Example of surface synaptic AMPARs cluster (GluA Bsn). Scale bar: 5 μm. Quantification of the surface synaptic AMPARs (GluA Bsn) normalized to the total number of Bsn shows a statistically significant increase after TTX or PTX3 exposure (Ctr = 1 ± 0.051, TTX = 1.512 ± 0.080, PTX3 = 1.294 ± 0.081, PTX3 heat‐inactivated = 0.959 ± 0.044; number of fields examined: 27, 36, 37, 22, 19, respectively; one‐way ANOVA, P

    Journal: The EMBO Journal

    Article Title: Pentraxin 3 regulates synaptic function by inducing AMPA receptor clustering via ECM remodeling and β1‐integrin

    doi: 10.15252/embj.201899529

    Figure Lengend Snippet: PTX3 increases excitatory glutamatergic neurotransmission by promoting AMPA receptors insertion at the synapse Representative mEPSC traces recorded from control and PTX3‐treated (1 μg/ml; 48 h) neurons. mEPSC frequency quantitation (Hz, Ctr = 0.618 ± 0.069; PTX3 = 1.991 ± 0.313; number of neurons: Ctr = 22, PTX3 = 16; three independent experiments, Mann–Whitney test, data are presented as a distribution plus mean ± SEM). mEPSC amplitude quantitation and cumulative probability distribution of mEPSC amplitudes (pA, Ctr = 12.76 ± 0.813; PTX3 = 16.07 ± 0.709. Number of neurons: Ctr = 22, PTX3 = 16; three independent experiments, Mann–Whitney test. Data are presented as a distribution plus mean ± SEM). Cumulative probability distributions are analyzed by Kolmogorov–Smirnov test. Representative images showing 14DIV neurons stained for surface AMPAR (GluA, green), the presynaptic protein Bassoon (blue), and tubulin (red) in the different tested conditions. Arrowheads point to postsynaptic GluA clusters. Inset: Example of surface synaptic AMPARs cluster (GluA Bsn). Scale bar: 5 μm. Quantification of the surface synaptic AMPARs (GluA Bsn) normalized to the total number of Bsn shows a statistically significant increase after TTX or PTX3 exposure (Ctr = 1 ± 0.051, TTX = 1.512 ± 0.080, PTX3 = 1.294 ± 0.081, PTX3 heat‐inactivated = 0.959 ± 0.044; number of fields examined: 27, 36, 37, 22, 19, respectively; one‐way ANOVA, P

    Article Snippet: The following antibodies were used: rabbit anti‐tubulin (1:100; T3526 Sigma‐Aldrich, Milan, Italy), guinea pig anti‐Bassoon (1:300; 141004, Synaptic Systems, Goettingen, Germany), mouse anti‐PSD95 (1:400; 75‐028, UC Davis/NIH NeuroMab Facility, CA), mouse anti‐gephyrin (1:500; 147021, Synaptic Systems, Goettingen, Germany), mouse anti‐beta III tubulin (1:400; G712A, Promega Corporation, Madison, USA), rabbit anti‐tubulin (1:80; Sigma‐Aldrich, Milan, Italy), rabbit anti‐aggrecan (1:200; AB1031, Millipore, Billerica, MA, USA), DAPI (1:5,000, Thermo Fisher).

    Techniques: Quantitation Assay, MANN-WHITNEY, Staining

    PTX3 does not modify structure and function of inhibitory synapses Representative images of 14DIV control and PTX3‐treated cultures stained for the presynaptic marker bassoon (blue), and the postsynaptic inhibitory protein gephyrin (green) and the microtubule protein tubulin (red). Scale bar: 5 μm. Quantification of synaptic density showing no differences either for postsynaptic marker (gephyrin) or for presynaptic marker (Bsn) or as a total number of synapses (geph Bsn) in control or PTX3‐treated neurons (gephyrin/μm, Ctr = 0.255 ± 0.014, PTX3 = 0.237 ± 0.016; Bsn/μm, Ctr = 0.239 ± 0.018, PTX3 = 0.191 ± 0.018; gephyrin Bsn/μm, ctr = 0.138 ± 0.010, PTX3 = 0.125 ± 0.014. Number of dendrites: Ctr = 71, PTX3 = 56; Mann–Whitney test. Three independent experiments, data are presented as mean ± SEM). Quantitative analysis of the mean size of gephyrin and bassoon puncta showing no differences in control and PTX3‐treated neurons (in μm 2 , gephyrin: Ctr = 0.109 ± 0.008; PTX3 = 0.095 ± 0.006; Bsn: Ctr = 0.107 ± 0.008, PTX3 = 0.099 ± 0.005, number of dendrites: Ctr = 53, PTX3 = 41; Mann–Whitney test; three independent experiments, data are presented as mean ± SEM). Representative traces of mIPSCs recorded from control and PTX3‐treated neurons. mIPSC frequency quantitation (Hz, Ctr = 1.464 ± 0.250; PTX3 = 1.792 ± 0.306. Number of neurons: Ctr = 24, PTX3 = 24; five independent experiments. Mann–Whitney test, data are presented as a distribution plus mean ± SEM). Cumulative probability plot and (inset) average of mIPSC amplitude (pA, Ctr = 15.43 ± 0.572; PTX3 = 16.58 ± 0.565. Number of neurons: Ctr = 24, PTX3 = 24; five independent experiments. Unpaired t ‐test, data are presented as a distribution, mean ± SEM and cumulative probability distribution of amplitudes analyzed with Kolmogorov–Smirnov test).

    Journal: The EMBO Journal

    Article Title: Pentraxin 3 regulates synaptic function by inducing AMPA receptor clustering via ECM remodeling and β1‐integrin

    doi: 10.15252/embj.201899529

    Figure Lengend Snippet: PTX3 does not modify structure and function of inhibitory synapses Representative images of 14DIV control and PTX3‐treated cultures stained for the presynaptic marker bassoon (blue), and the postsynaptic inhibitory protein gephyrin (green) and the microtubule protein tubulin (red). Scale bar: 5 μm. Quantification of synaptic density showing no differences either for postsynaptic marker (gephyrin) or for presynaptic marker (Bsn) or as a total number of synapses (geph Bsn) in control or PTX3‐treated neurons (gephyrin/μm, Ctr = 0.255 ± 0.014, PTX3 = 0.237 ± 0.016; Bsn/μm, Ctr = 0.239 ± 0.018, PTX3 = 0.191 ± 0.018; gephyrin Bsn/μm, ctr = 0.138 ± 0.010, PTX3 = 0.125 ± 0.014. Number of dendrites: Ctr = 71, PTX3 = 56; Mann–Whitney test. Three independent experiments, data are presented as mean ± SEM). Quantitative analysis of the mean size of gephyrin and bassoon puncta showing no differences in control and PTX3‐treated neurons (in μm 2 , gephyrin: Ctr = 0.109 ± 0.008; PTX3 = 0.095 ± 0.006; Bsn: Ctr = 0.107 ± 0.008, PTX3 = 0.099 ± 0.005, number of dendrites: Ctr = 53, PTX3 = 41; Mann–Whitney test; three independent experiments, data are presented as mean ± SEM). Representative traces of mIPSCs recorded from control and PTX3‐treated neurons. mIPSC frequency quantitation (Hz, Ctr = 1.464 ± 0.250; PTX3 = 1.792 ± 0.306. Number of neurons: Ctr = 24, PTX3 = 24; five independent experiments. Mann–Whitney test, data are presented as a distribution plus mean ± SEM). Cumulative probability plot and (inset) average of mIPSC amplitude (pA, Ctr = 15.43 ± 0.572; PTX3 = 16.58 ± 0.565. Number of neurons: Ctr = 24, PTX3 = 24; five independent experiments. Unpaired t ‐test, data are presented as a distribution, mean ± SEM and cumulative probability distribution of amplitudes analyzed with Kolmogorov–Smirnov test).

    Article Snippet: The following antibodies were used: rabbit anti‐tubulin (1:100; T3526 Sigma‐Aldrich, Milan, Italy), guinea pig anti‐Bassoon (1:300; 141004, Synaptic Systems, Goettingen, Germany), mouse anti‐PSD95 (1:400; 75‐028, UC Davis/NIH NeuroMab Facility, CA), mouse anti‐gephyrin (1:500; 147021, Synaptic Systems, Goettingen, Germany), mouse anti‐beta III tubulin (1:400; G712A, Promega Corporation, Madison, USA), rabbit anti‐tubulin (1:80; Sigma‐Aldrich, Milan, Italy), rabbit anti‐aggrecan (1:200; AB1031, Millipore, Billerica, MA, USA), DAPI (1:5,000, Thermo Fisher).

    Techniques: Staining, Marker, MANN-WHITNEY, Quantitation Assay

    CGRP + and β III tubulin fibers in the epidermis and hair follicles of wounded skin. The panels show photomicrographs of CGRP (A1, B1, E1, F1) and β III tubulin (βTub; A2, B2, E2, F2) immunostaining of skin sections of control (A and E) and treated rats (B and F) at 32 h postwounding. The βTub + fibers were abundantly distributed throughout the epidermis of control rats, while the epidermis of treated rats was practically devoid of this type of fibers. Note that CGRP + fibers in the treated rats were mainly located in the dermis and not associated with epithelium (B). epi, epidermis; der, dermis; sg, sebaceous gland; b, bulge; hf, hair follicle. Scale bar = 50 µm.

    Journal: PLoS ONE

    Article Title: Modulatory Role of Sensory Innervation on Hair Follicle Stem Cell Progeny during Wound Healing of the Rat Skin

    doi: 10.1371/journal.pone.0036421

    Figure Lengend Snippet: CGRP + and β III tubulin fibers in the epidermis and hair follicles of wounded skin. The panels show photomicrographs of CGRP (A1, B1, E1, F1) and β III tubulin (βTub; A2, B2, E2, F2) immunostaining of skin sections of control (A and E) and treated rats (B and F) at 32 h postwounding. The βTub + fibers were abundantly distributed throughout the epidermis of control rats, while the epidermis of treated rats was practically devoid of this type of fibers. Note that CGRP + fibers in the treated rats were mainly located in the dermis and not associated with epithelium (B). epi, epidermis; der, dermis; sg, sebaceous gland; b, bulge; hf, hair follicle. Scale bar = 50 µm.

    Article Snippet: In additional experiments, double immunofluorescence was performed with mouse anti-neural III beta-tubulin (1∶500, Promega, Madison, USA).

    Techniques: Immunostaining