Journal: The Journal of Experimental Medicine

Article Title: Follicular dendritic cells help establish follicle identity and promote B cell retention in germinal centers

doi: 10.1084/jem.20111449

Figure Lengend Snippet: DTx-mediated selective FDC ablation disrupts B cell follicle architecture despite retention of CXCL13. Unimmunized CD21-DTR chimeras were control (Ctrl) or DTx treated 2 d before analysis of spleen (spl) and pLN sections. (A) Immunofluorescence analysis for BP3 + stromal cells, CD35 + FDCs, and IgD + follicular B cells. (B) In situ hybridization for Cxcl13 mRNA with serial sections stained for B220 and CD35 or for Cxcl13 and CD35. (C) Quantitative RT-PCR analysis for Cxcl13 , Cr1 (CD35), Mfge8 , and Tnsf13B (BAFF) mRNA in pLNs, spleen, and mLNs, normalized to Hprt mRNA. Data are representative of four experiments (mean ± SEM). Statistical analysis was performed with the two-tailed unpaired Student’s t test. *, P < 0.05; n.s., not significant. (D) Immunohistochemical analysis for CXCL13 and CD3 or B220 in serial sections of spleen and pLNs. Data in A, B, and D are representative of at least three experiments (at least two mice of each type per experiment). Bars: (A [left] and B [left and middle]) 200 µm; (A and D, right) 100 µm; (B [right] and D [left]) 50 µm.

Article Snippet: Cryosections of 7 μm were fixed and stained immunohistochemically as previously described ( ) with the following first antibodies: PE-conjugated anti-IgD (11-26c.2a; BD), biotin-conjugated anti-CD35 (8C12; BD), rat anti–mouse fibroblast (ER-TR7; Novus Biologicals), rabbit anti–mouse Collagen IV (Abcam), rat anti–mouse B220 (RA-6B2; BD), biotin-conjugated anti–T/B cell activation antigen or Ly77 (GL7; BD), biotin conjugated anti–mouse CD3e (145-2C11; BD), goat anti–mouse CXCL13 (R&D Systems), goat anti–mouse CCL21 (R&D Systems), rat anti-CD169 (Ser4; P. Crocker, University of Glasgow, Glasgow, Scotland, UK), rat anti–mouse madcam-1 (MECA-367; BD), rat anti–mouse FDC (FDC-M1; BD), and FITC-conjugated rat anti–mouse CD16/32 (FcγRII/III; UCSF Hybridoma Core).

Techniques: Control, Immunofluorescence, In Situ Hybridization, Staining, Quantitative RT-PCR, Two Tailed Test, Immunohistochemical staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Selectin ligand-independent priming and maintenance of T cell immunity during airborne tuberculosis.

doi: 10.4049/jimmunol.176.2.1131

Figure Lengend Snippet: FIGURE 9. M. tuberculosis-infection induces CXCL13, CCL19, and CCL21 expression in lungs from FucT-deficient mice. C57BL/6 (black bars), FucT-IV/

Article Snippet: For immunohistochemical detection of chemokine production in lungs from infected mice, tissue sections were incubated overnight at 4°C with primary goat Abs to mouse CXCL13 (R&D Systems) or CCL21 (R&D Systems), followed by incubation with biotin-SP-AffiniPure mouse anti-goat IgG (Jackson ImmunoResearch Laboratories).

Techniques: Infection, Expressing

Journal: EBioMedicine

Article Title: CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2020.103097

Figure Lengend Snippet: Motor neurons strongly activate CXCL13 in the spinal cord of fast mSOD1 mice, and the chemokine is released in the CSF during the disease progression. (a) Cxcl13 mRNA fold change levels from laser captured MNs of C57-mSOD1 and 129Sv-mSOD1 mice. mRNA levels are expressed as mean fold change ratio (± SEM) between C57-mSOD1 versus C57Ntg ( n = 4) mice and between 129Sv-mSOD1 ( n = 4) versus 129SvNTg ( n = 4) mice. ( b, c ) Real-time PCR for CXCL13 transcript in the lumbar spinal cord of (b) 129Sv-mSOD1 and (c) C57-mSOD1 mice. Data are normalised to β-actin and expressed as the mean ± SEM fold-change ratio between C57-mSOD1 and 129Sv-mSOD1 mice compared to respective age-matched Ntg littermates. ⁎⁎ P < 0.01; ⁎⁎⁎⁎ P < 0.0001 (Vs. Ntg) by unpaired t -test. ( d-g ) Fast progressing mice showed a striking activation of CXCL13 in the spinal cord at the disease onset, although the chemokine is upregulated in both (e) 129Sv-mSOD1 and (g) C57-mSOD1 mice compared to respective (d, f) Ntg littermates; scale bar, 50 μm. ( h ) Representative co-localization of CXCL13 (green) with the neuronal marker (Neurotrace; NT; red) in the ventral horn of the spinal cord of fast-progressing mice; scale bar: 20 μm. ( i ) Confocal representative image showing CXCL13 staining in correspondence of efferent motor axons (white arrowheads); scale bar: 20 μm. ( j ) Representative co-localization of CXCL13 (green) with the microglia marker (CD68) in the ventral horn of the spinal cord of fast-progressing mice. ( k ) Confocal image of the ventral portion of the spinal cord showing the lack of co-localisation of CXCL13 (green) with the astrocyte marker GFAP (red). ( l - o ) CXCL13 levels (pg/mL) in the CSF and serum of (l, n) 129Sv-mSOD1 and (m, o) C57-mSOD1 mice during the disease progression. Data are expressed as the mean ± SEM. * P < 0.05; ** P < 0.001; **** P < 0.0001 (Vs. Ntg); ° P < 0.05; °° P < 0.01; (between groups) by one-way ANOVA with Tukey's post-analysis.

Article Snippet: Antigen retrieval was done in citrate buffer (Dako), and Alexa 647-Tyramide amplification (Perkin Elmer) or peroxidase-diaminobenzidine (DAB) reaction were used for detecting CXCL13 using anti-human rabbit CXCL13 (1:100; Bioss Antibodies); anti-phospho TD43(1:5000; Cosmo Bio Co., LTD); anti-CXCR5 (1:100; Abcam) and Neuro-Trace conjugated with Alexa-594 (1:500; Invitrogen) were used in immunofluorescence for detecting pTD43 inclusions and neuronal cells, respectively.

Techniques: Real-time Polymerase Chain Reaction, Activation Assay, Marker, Staining

Journal: EBioMedicine

Article Title: CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2020.103097

Figure Lengend Snippet: CXCR5 is upregulated in the motor neurons of mSOD1 mice during the disease progression. (a) Real-time PCR for CXCL13 transcript in the lumbar spinal cord of 129Sv-mSOD1 mice. Data are normalised to β-actin and expressed as the mean ± SEM fold-change ratio between 129Sv-mSOD1 mice compared to respective age-matched Ntg littermates. * P < 0.05; ⁎⁎ P < 0.01 (Vs. Ntg) by one-way ANOVA with Tukey's post-analysis. ( b ) Representative co-localization of CXCR5 (green) with NT (purple) but not with GFAP (red) in the ventral horn of the spinal cord of fast-progressing mice; scale bar: 20 μm. ( c ) Confocal representative images showing the progressive increase in CXCR5 staining in the lumbar spinal cord of 129Sv-mSOD1 mice.

Article Snippet: Antigen retrieval was done in citrate buffer (Dako), and Alexa 647-Tyramide amplification (Perkin Elmer) or peroxidase-diaminobenzidine (DAB) reaction were used for detecting CXCL13 using anti-human rabbit CXCL13 (1:100; Bioss Antibodies); anti-phospho TD43(1:5000; Cosmo Bio Co., LTD); anti-CXCR5 (1:100; Abcam) and Neuro-Trace conjugated with Alexa-594 (1:500; Invitrogen) were used in immunofluorescence for detecting pTD43 inclusions and neuronal cells, respectively.

Techniques: Real-time Polymerase Chain Reaction, Staining

Journal: EBioMedicine

Article Title: CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2020.103097

Figure Lengend Snippet: The intra-cerebroventricular neutralisation of CXCL13 accelerates the symptom onset and reduced the survival of mSOD1 mice. (a) Body weight loss and (b) Paw Grip Endurance (PaGE) test for mAb-αCXCL13- ( n = 14) and IgG- treated ( n = 17) 129Sv-mSOD1 mice. Data are reported as mean ± SEM for each time point. * P < 0.05; ⁎⁎⁎⁎ P < 0.0001 by repeated-measures ANOVA with Sidak's post-analysis. ( c ) mAb-αCXCL13-treated mice show an earlier impairment in the extension reflex of hindlimbs * P < 0.05; ⁎⁎ P < 0.01; ⁎⁎⁎ P < 0.001 ⁎⁎⁎⁎ P < 0.0001 by repeated-measures ANOVA with Sidak's post-analysis. ( d ) mAb-αCXCL13-treated mice have an earlier onset of motor impairment than IgG- treated mice. P < 0.0055 by Mantel-Cox log-rank test. ( e ) mAb-αCXCL13-treated 129Sv-mSOD1 mice display a reduced survival in respect IgG- treated 129Sv-mSOD1 mice. P < 0.0303 by Mantel-Cox log-rank test. ( f ) Percentage of surviving mAb α-CXCL13 treated mice and controls at different time points (days) after the end of the treatment (16 weeks).

Article Snippet: Antigen retrieval was done in citrate buffer (Dako), and Alexa 647-Tyramide amplification (Perkin Elmer) or peroxidase-diaminobenzidine (DAB) reaction were used for detecting CXCL13 using anti-human rabbit CXCL13 (1:100; Bioss Antibodies); anti-phospho TD43(1:5000; Cosmo Bio Co., LTD); anti-CXCR5 (1:100; Abcam) and Neuro-Trace conjugated with Alexa-594 (1:500; Invitrogen) were used in immunofluorescence for detecting pTD43 inclusions and neuronal cells, respectively.

Techniques:

Journal: EBioMedicine

Article Title: CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2020.103097

Figure Lengend Snippet: Intra-cerebroventricular neutralisation of CXCL13 exacerbated motor neuron impairment, and astrocytosis in the lumbar spinal cord of mSOD1 mice. (a, b) Real-time PCR for (a) Cxcl13 and (b) Cxcr5 transcripts in the lumbar spinal cord of mAb-αCXCL13- and IgG- treated 129Sv-mSOD1 mice compared to Ntg littermates at 16 weeks of age. Data are normalised to β-actin and expressed as the mean ± SEM fold-change ratio between mAb-αCXCL13- and IgG- treated 129Sv-mSOD1 mice and controls. ⁎⁎⁎⁎ P < 0.0001 (Vs Ntg); ° P <0.05 by one-way ANOVA with Tukey's post-analysis. ( c - f ) Representative Immunoblot images and densitometric analysis of (c, d) ChAT (c, e) GFAP (c, f) P-AKT expression in spinal cord extracts from mAb-αCXCL13- and IgG- treated 129Sv-mSOD1 mice compared to Ntg littermates at 16 weeks of age. Data are reported as percentages of the relative Ntg (mean ± SEM). * P < 0.05; ⁎⁎ P < 0.01: ⁎⁎⁎ P < 0.001; ⁎⁎⁎⁎ P < 0.0001 (Vs. Ntg); ° P < 0.05 (between groups) by one-way ANOVA with Tukey's post-analysis.

Article Snippet: Antigen retrieval was done in citrate buffer (Dako), and Alexa 647-Tyramide amplification (Perkin Elmer) or peroxidase-diaminobenzidine (DAB) reaction were used for detecting CXCL13 using anti-human rabbit CXCL13 (1:100; Bioss Antibodies); anti-phospho TD43(1:5000; Cosmo Bio Co., LTD); anti-CXCR5 (1:100; Abcam) and Neuro-Trace conjugated with Alexa-594 (1:500; Invitrogen) were used in immunofluorescence for detecting pTD43 inclusions and neuronal cells, respectively.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing

Journal: EBioMedicine

Article Title: CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2020.103097

Figure Lengend Snippet: Intra-cerebroventricular neutralisation of CXCL13 exacerbated the denervation atrophy of hindlimb skeletal muscles of mSOD1 mice. (a, b) Confocal micrographs showing higher levels of CXCL13 (red) in the sciatic nerve of 129Sv-mSDO1 mice at disease onset and co-localisation with the axonal marker NF200 (green); scale bars 200 μm. ( c-e ) Confocal micrographs showing higher levels of CXCR5 (green) in the sciatic nerve of 129Sv-mSDO1 mice than Ntg mice at disease onset; scale bar: 100 μm. CXCR5 is present in the bundles of Schwann cells (S100β, red), as evidenced by ( e ) the volume view of e. ( f, g ) Confocal micrographs showing the colocalisation of CXCL13 (red), and CXCR5 (green) in correspondence of peripheral axons (Smi32; purple) in the sciatic nerves of 129Sv-mSDO1 mice at the disease onset Scale bar: (f) 50 μm; (g) 10 μm. ( k ) Percent muscle atrophy (muscle wasting) calculated by measuring of the tibialis anterior muscle weight of mAb-αCXCL13- and IgG-treated 129Sv-mice at 16 weeks and endstage compared to relative Ntg littermates. Data are presented as mean ± SEM. ° P < 0.05 by unpaired t -test. ( h-j ) Analysis of muscle denervation on tibialis anterior (TA) muscle of both mAb-αCXCL13-, IgG-treated 129Sv-mice and Ntg littermates at 16 weeks of age. α-Bungarotoxin (BTX, green) was used to identify the postsynaptic domain, synaptic vesicle glycoprotein 2A (SV2, red) + neurofilaments (2H3, red) were used to identify presynaptic terminals. Scale bar 20 μm. ( l ) For each mouse group, the percentage of occupied endplates was calculated. Data are reported as mean ± SEM. ⁎⁎⁎⁎ P < 0.0001 (Vs. Ntg) ° P < 0.05 (between groups) by one-way ANOVA with Tukey's post-analysis.

Article Snippet: Antigen retrieval was done in citrate buffer (Dako), and Alexa 647-Tyramide amplification (Perkin Elmer) or peroxidase-diaminobenzidine (DAB) reaction were used for detecting CXCL13 using anti-human rabbit CXCL13 (1:100; Bioss Antibodies); anti-phospho TD43(1:5000; Cosmo Bio Co., LTD); anti-CXCR5 (1:100; Abcam) and Neuro-Trace conjugated with Alexa-594 (1:500; Invitrogen) were used in immunofluorescence for detecting pTD43 inclusions and neuronal cells, respectively.

Techniques: Marker

Journal: EBioMedicine

Article Title: CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2020.103097

Figure Lengend Snippet: Silencing of CXCL13 expression heightened MN loss and astrocytosis in non-transgenic and mSOD1 primary co-cultures. (a-c) Confocal micrographs showing the co-localisation of CXCL13 (red) with (a, b) the neuronal marker Smi32 (green) and with (c) the microglia marker Iba1; Scale bar, (a, b): 100 μm; (c): 200 μm. ( d ) CXCL13 concentration (pg/mL) in the conditional media of shRNA-CXCl13- and shRNA- scramble (SCR)-treated or untreated (UT) mSOD1 and Ntg co-cultured with mSOD1 and Ntg microglia/astrocytes, respectively. Data are expressed as the mean ± SEM from three independent experiments for each experimental group. * P < 0.05; ⁎⁎ P < 0.001; ⁎⁎⁎ P < 0.001 Kruskall-Wallis ANOVA with uncorrected Dunn's post analysis. (e) Representative SMI-32 immunostaining images (white) of shRNA-CXCl13- and shRNA-SCR-treated mSOD1 and Ntg motor neurons co-cultured with mSOD1 and Ntg microglia/astrocytes, respectively; scale bar 200 μm. ( f ) Cell count analysis showing greater MN loss in both shRNA-CXCl13 treated mSOD1 and Ntg co-cultures than or shRNA-SCR- treated or untreated (UT) co-cultures. Data are reported as mean ± SEM of the No. fields analysed from three independent experiments for each experimental group. * P < 0.05; ⁎⁎ P < 0.001; ⁎⁎⁎ P < 0.001; ⁎⁎⁎⁎ P < 0.0001 by Kruskall-Wallis ANOVA with uncorrected Dunn's post analysis. ( g ) Representative GFAP immunostaining images (green) of shRNA-CXCl13- and shRNA-SCR-treated mSOD1 and Ntg astrocytes co-cultured with mSOD1and Ntg motor neurons/microglia, respectively; scale bar 100 μm. ( h ) Cell Area Fraction (percentage) analysis showing greater astrocytosis in shRNA-CXCl13 treated mSOD1 co-cultures than shRNA-SCR-treated or UT mSOD1 co-cultures. Data are reported as mean ± SEM of the No. fields analysed from three independent experiments for each experimental group. * P < 0.05; ⁎⁎ P < 0.01; ⁎⁎⁎ P < 0.001 ⁎⁎⁎⁎ P < 0.0001 by Kruskall-Wallis ANOVA with uncorrected Dunn's post analysis. ( i ) Representative Iba1 immunostaining images (red) of shRNA-CXCl13- and shRNA-SCR-treated mSOD1 and Ntg microglia co-cultured respectively with mSOD1and Ntg motor neurons/astrocytes; scale bar 100 μm. ( j ) Morphometric parameter of Iba1-positive microglia showing higher activation (mean microglial cell area) of shRNA-CXCl13-treated mSOD1 microglia than shRNA-SCR- treated or UT mSOD1 co-cultures. Data are reported as mean ± SEM of the No. fields analysed from three independent experiments for each experimental group. * P < 0.05; ⁎⁎ P < 0.001; ⁎⁎⁎ P < 0.001; ⁎⁎⁎⁎ P < 0.0001 by Kruskall-Wallis ANOVA with uncorrected Dunn's post analysis.

Article Snippet: Antigen retrieval was done in citrate buffer (Dako), and Alexa 647-Tyramide amplification (Perkin Elmer) or peroxidase-diaminobenzidine (DAB) reaction were used for detecting CXCL13 using anti-human rabbit CXCL13 (1:100; Bioss Antibodies); anti-phospho TD43(1:5000; Cosmo Bio Co., LTD); anti-CXCR5 (1:100; Abcam) and Neuro-Trace conjugated with Alexa-594 (1:500; Invitrogen) were used in immunofluorescence for detecting pTD43 inclusions and neuronal cells, respectively.

Techniques: Expressing, Transgenic Assay, Marker, Concentration Assay, shRNA, Cell Culture, Immunostaining, Cell Counting, Activation Assay

Journal: EBioMedicine

Article Title: CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2020.103097

Figure Lengend Snippet: CXCL13 protects motor neurons under acute and chronic treatment. (a, g) Representative SMI-32 immunostaining images (white) of rCXCL13- and rCXCL13+ mAb-αCXCL13- treated co-cultures in the presence (a) or (g) absence of LPS stimulus; scale bar 200 μm. (d, j ) Cell count analysis showing the protective effect of rCXCL13 in the presence (d) or (j) absence of LPS stimulus compared to CXCL13+ mAb-αCXCL13-treated co-cultures. Data are reported as the percentage of UT co-cultures (mean ± SEM of the No. fields analysed from three independent experiments for each experimental group). * P < 0.05; ⁎⁎ P < 0.01 (Vs. UT); °°° P < 0.01 (between groups) by one-way ANOVA with Tukey's post-analysis. (b, h) Representative GFAP immunostaining images (blue) of rCXCL13- and rCXCL13+ mAb-αCXCL13- treated co-cultures in the presence (b) or (h) absence of LPS stimulus; scale bar 100 μm. ( e, k ) Fluorescence Integrated Density (Int_Den) analysis showing greater astrocytosis in rCXCL13 + mAb-αCXCL13-treated co-cultures in the presence (e) or (k) absence of LPS stimulus compared to rCXCL13-treated or UT co-cultures. Data are reported as mean ± SEM of the No. fields analysed from three independent experiments for each experimental group. * P < 0.05; ⁎⁎⁎ P < 0.001; ⁎⁎⁎⁎ P < 0.0001 (Vs. UT); °°°° P < 0.01 (between groups) by one-way ANOVA with Tukey's post-analysis. ( c, i ) Representative Iba1 immunostaining images (green) of rCXCL13- and rCXCL13+ mAb-αCXCL13- treated co-cultures in the presence (c) or (i) absence of LPS stimulus; scale bar 100 μm. ( f, l ) Morphometric parameter of Iba1-positive microglia showing no difference in activation (mean microglial cell area) between the experimental groups. Data are reported as mean ± SEM of the No. fields analysed from three independent experiments for each experimental group. * P < 0.05; ⁎⁎ P < 0.01; ⁎⁎⁎⁎ P < 0.0001 (Vs. UT) by one-way ANOVA with Tukey's post-analysis.

Article Snippet: Antigen retrieval was done in citrate buffer (Dako), and Alexa 647-Tyramide amplification (Perkin Elmer) or peroxidase-diaminobenzidine (DAB) reaction were used for detecting CXCL13 using anti-human rabbit CXCL13 (1:100; Bioss Antibodies); anti-phospho TD43(1:5000; Cosmo Bio Co., LTD); anti-CXCR5 (1:100; Abcam) and Neuro-Trace conjugated with Alexa-594 (1:500; Invitrogen) were used in immunofluorescence for detecting pTD43 inclusions and neuronal cells, respectively.

Techniques: Immunostaining, Cell Counting, Fluorescence, Activation Assay

Journal: EBioMedicine

Article Title: CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2020.103097

Figure Lengend Snippet: CXCL13 is upregulated in the motor neurons of the spinal cord of familial and sporadic ALS patients. (a-l) Representative confocal images of immunofluorescence staining for pTDP43, CXCL13, and Neurotrace (neuronal marker) in the post-mortem lumbar spinal cord of (e-l) n = 4 sporadic (sALS) and n = 4 familial (fALS) ALS patients showing the upregulation of the CXCL13 staining in motor neurons compared to (a-d) n = 4 controls; Scale bar: 100 μm; Inset: 50 μm.

Article Snippet: Antigen retrieval was done in citrate buffer (Dako), and Alexa 647-Tyramide amplification (Perkin Elmer) or peroxidase-diaminobenzidine (DAB) reaction were used for detecting CXCL13 using anti-human rabbit CXCL13 (1:100; Bioss Antibodies); anti-phospho TD43(1:5000; Cosmo Bio Co., LTD); anti-CXCR5 (1:100; Abcam) and Neuro-Trace conjugated with Alexa-594 (1:500; Invitrogen) were used in immunofluorescence for detecting pTD43 inclusions and neuronal cells, respectively.

Techniques: Immunofluorescence, Staining, Marker

Journal: EBioMedicine

Article Title: CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2020.103097

Figure Lengend Snippet: CXCL13 has a low concentration in the CSF of ALS patients (a, b) CXCL13 concentration (pg/mL) in the CSF of (a) total ALS patients and (b) sub-grouped by disease onset and normative controls. ( c ) CXCL13 concentration (pg/mL) in the CSF of ALS patients and MS patients. Data are reported as mean ± SEM. P < 0.05 by Mann-Whitney test. ( d ) ROC curve and analysis of the area under the curve (AUC) were used to find the discriminatory power of CXCL13 CSF levels between ALS and MS patients. A 95% Confidence Interval (CI) was used and results are reported as percentage.

Article Snippet: Antigen retrieval was done in citrate buffer (Dako), and Alexa 647-Tyramide amplification (Perkin Elmer) or peroxidase-diaminobenzidine (DAB) reaction were used for detecting CXCL13 using anti-human rabbit CXCL13 (1:100; Bioss Antibodies); anti-phospho TD43(1:5000; Cosmo Bio Co., LTD); anti-CXCR5 (1:100; Abcam) and Neuro-Trace conjugated with Alexa-594 (1:500; Invitrogen) were used in immunofluorescence for detecting pTD43 inclusions and neuronal cells, respectively.

Techniques: Concentration Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: A Standardized Analysis of Tertiary Lymphoid Structures in Human Melanoma: Disease Progression- and Tumor Site-Associated Changes With Germinal Center Alteration

doi: 10.3389/fimmu.2021.675146

Figure Lengend Snippet: Detection of different TLS phenotypes by 7-color multiplex immunohistochemistry in human melanoma tissues. Examples for (A) early TLS: dense CD20 + lymphocyte aggregates with CXCL13 + cells and the presence of some interspersed CD4 + T cells; (B) primary follicular TLS: CD20 + lymphocyte aggregates interspersed with CD4 + T cells and the presence of a CD21 + but CD23 - dendritic network, surrounded by CXCL13 expressing cells; (C) secondary follicular TLS with a BCL6 - germinal center: CD21 + and CD23 + dendritic networks within CD20 + lymphocyte aggregates with interspersed CD4 + T cells, surrounded by CXCL13 expressing cells. No accessory expression of BCL6 in lymphatic cells. Germinal centers are identifiable in routine light microscopy (see Pathology View, bottom left). An example for a BCL6 + secondary follicular TLS is given in Figure 5A . Images for each of the individual markers and their composites (for clarity without DAPI staining) are shown, together with the corresponding Pathology View (respective bottom left). Scale bars represent 100 µm. (D) Spatial annotation of tumor areas. Left: Definition of the invasive tumor front (red, 2) and the respective intra- and extratumoral compartments (brown, 1 and yellow, 3, respectively) in a tissue section of a human melanoma lymph node metastasis. Extratumoral compartments include site-specific and adipose tissue (yellow). Right: Allocation of intra- (brown/black) and extratumoral perimeters (green) of increasing radiuses (1 – max. 6 mm) drawn within and around the invasive tumor front. Counts and area of TLS were referred to the tissue area (yellow) within the respective intra- and extratumoral perimeters. TLS, tertiary lymphoid structures.

Article Snippet: First, each of the following antibodies was established on four-micrometer sections from FFPE tissue of human tonsil: CD20 (mouse monoclonal IgG2a, clone L26, 1:2000, Agilent, M0755), CD4 (mouse monoclonal IgG1, clone 4B12, 1:500, Agilent, M7310), CXCL13 (rabbit polyclonal IgG, 1:1000, Proteintech, 10927-1-AP), CD21 (rabbit polyclonal IgG, 1:3600, Proteintech, 24374-1-AP), CD23 (rabbit monoclonal IgG, clone SP23, 1:900, Novus Biologicals, NB120-16702) and BCL6 (mouse monoclonal IgG1, clone 1E6B1, 1:24000, Proteintech, 66340-1-Ig).

Techniques: Multiplex Assay, Immunohistochemistry, Expressing, Light Microscopy, Staining

Journal: Frontiers in Immunology

Article Title: A Standardized Analysis of Tertiary Lymphoid Structures in Human Melanoma: Disease Progression- and Tumor Site-Associated Changes With Germinal Center Alteration

doi: 10.3389/fimmu.2021.675146

Figure Lengend Snippet: Comparison of large BCL6 + secondary follicular TLS in human melanoma to tonsil tissue. (A) Melanoma: CD20 + lymphocyte aggregates with a germinal center surrounded by CXCL13 expressing cells. A CD21 + and CD23 + FDC network covers the complete germinal center area with randomly interspersed CD4 + T cells and BCL6 + cells. No detectable germinal center polarity, no identifiable light and dark zone areas in routine light microscopy (see Pathology View, bottom left). (B) Close up of BCL6 + CD20 + B cells and BCL6 + CD4 + T cells from a BCL6 + secondary follicular TLS in human melanoma. (C) Tonsil: note canonical germinal center polarity with polarized spatial distribution of the CD21 + and CD23 + FDC network together with CD4 + T cells (arrowheads) and a complementary localization of BCL6 + cells (arrow). Identifiable light (arrowhead) and dark zone (arrow) areas in routine light microscopy (see Pathology View, bottom left). (D) Tonsil (upper row): polarized distribution of BCL6 + , Ki67 + (arrows) and BCL6 + Ki67 + cells in germinal centers with canonical germinal center polarity and the presence of light (arrowhead) and dark zone (arrow) areas in routine light microscopy (see Pathology View, left). Melanoma (lower row): note decreased numbers and absence of polarized distribution of BCL6 + , Ki67 + and BCL6 + Ki67 + cells in melanoma germinal centers. Lack of canonical germinal center polarity and absence of light and dark zone areas in routine light microscopy (see Pathology View, left). Images for each of the individual markers and their composites (for clarity without DAPI staining) are shown, together with the corresponding Pathology View (respective (bottom) left). Scale bars represent 100 µm (A, C, D) and 5 µm (B) .

Article Snippet: First, each of the following antibodies was established on four-micrometer sections from FFPE tissue of human tonsil: CD20 (mouse monoclonal IgG2a, clone L26, 1:2000, Agilent, M0755), CD4 (mouse monoclonal IgG1, clone 4B12, 1:500, Agilent, M7310), CXCL13 (rabbit polyclonal IgG, 1:1000, Proteintech, 10927-1-AP), CD21 (rabbit polyclonal IgG, 1:3600, Proteintech, 24374-1-AP), CD23 (rabbit monoclonal IgG, clone SP23, 1:900, Novus Biologicals, NB120-16702) and BCL6 (mouse monoclonal IgG1, clone 1E6B1, 1:24000, Proteintech, 66340-1-Ig).

Techniques: Comparison, Expressing, Light Microscopy, Staining

Journal: European journal of immunology

Article Title: Systematic microanatomical analysis of CXCL13 and CCL21 in situ production and progressive lymphoid organization in rheumatoid synovitis.

doi: 10.1002/eji.200425830

Figure Lengend Snippet: Fig. 3. CXCL13 and CCL21 production and lymphoid organization. IHC for CD3, CD20, CD21, CXCL13 and CCL21 (red) and ISH for CXCL13 and CCL21 (black) were performed on consecutive sections of RA synovium. Representative aggregate areas featuring increasing levels of structural organization, with intermixed T and B cells (A–C), T-B cell compartmentalization (H–J) and T-B cell compartmentalization with centrally located CD21+ FDC networks (O–Q), are illustrated. CXCL13 and CCL21 protein and mRNA are present inside a follicular Grade 3 aggregate (R, S and T, U) and in less organized aggregate areas (K, L and D, E for CXCL13 and M, N for CCL21). Inside the follicular aggregate, CXCL13-producing cells (R, S) are found inside and outside the CD21+ FDC network-rich area (Q), with a predominant localization in the B cell distribution zone (P). Note the colocalization between CCL21 (T, U) and the T cell-rich compartment (O).

Article Snippet: The following primary Ab were used: rabbit anti-human CD3 polyclonal (Ig, A0452, DAKO), mouse antihuman CD20 (IgG2a, clone L26, DAKO), mouse anti-human CD21 (IgG1, clone 1F8, DAKO), mouse anti-human CD35 (IgG2b, clone RLB25, Novocastra), mouse anti-human CXCL13 (IgG1, clone 53610, R&D Systems), goat anti-human CXCL13 polyclonal (IgG, AF801, R&D Systems), goat anti-human CCL21 polyclonal (IgG, AF366, R&D Systems), mouse antihuman CD31 (IgG1, clone 1A10, Novocastra) and rat antihuman/mouse PNAd (IgM, clone MECA 79, PharMingen).

Techniques:

Journal: European journal of immunology

Article Title: Systematic microanatomical analysis of CXCL13 and CCL21 in situ production and progressive lymphoid organization in rheumatoid synovitis.

doi: 10.1002/eji.200425830

Figure Lengend Snippet: Fig. 4. CXCL13 production and histomorphological grading. Protein and mRNA expression of CXCL13 were detected by IHC and ISH, respectively, on consecutive sections of RA synovium (positive cells in black). CXCL13-expressing cells were localized in sublining areas characterized by a high density of mono- nuclear inflammatory infiltrate, with a strong association with large cellular aggregates (A, B). Few CXCL13-producing cells were present in areas featuring a high density of diffuse infiltrate and small-sized aggregates (C, D; higher magnifica- tion E, F). Representative images of Grade 1 (G, H), Grade 2 (I, J) and Grade 3 aggregates (K, L) are shown.

Article Snippet: The following primary Ab were used: rabbit anti-human CD3 polyclonal (Ig, A0452, DAKO), mouse antihuman CD20 (IgG2a, clone L26, DAKO), mouse anti-human CD21 (IgG1, clone 1F8, DAKO), mouse anti-human CD35 (IgG2b, clone RLB25, Novocastra), mouse anti-human CXCL13 (IgG1, clone 53610, R&D Systems), goat anti-human CXCL13 polyclonal (IgG, AF801, R&D Systems), goat anti-human CCL21 polyclonal (IgG, AF366, R&D Systems), mouse antihuman CD31 (IgG1, clone 1A10, Novocastra) and rat antihuman/mouse PNAd (IgM, clone MECA 79, PharMingen).

Techniques: Expressing

Journal: European journal of immunology

Article Title: Systematic microanatomical analysis of CXCL13 and CCL21 in situ production and progressive lymphoid organization in rheumatoid synovitis.

doi: 10.1002/eji.200425830

Figure Lengend Snippet: Fig. 5. CXCL13 production and the synovial vascular system. Expression of CXCL13 (A, C, E), CD31 (D) and PNAd (F) was detected in sections of RA synovium by IHC (positive cells in black). mRNA expression of CXCL13 was determined by ISH (B) (black). Synovial vascular structures (arrowheads) were not recognized as CXCL13 protein-producing sites as determined by the absence of detectable levels of endothelial CXCL13 protein production (A for CXCL13 protein and B for CXCL13 mRNA). This observation was confirmed by the analysis of CXCL13 protein expression in CD31+ vessels and PNAd+ HEV (arrowheads in C, D and E, F). CXCL13 mRNA was not detected in human lymph node HEV (arrowheads in G).

Article Snippet: The following primary Ab were used: rabbit anti-human CD3 polyclonal (Ig, A0452, DAKO), mouse antihuman CD20 (IgG2a, clone L26, DAKO), mouse anti-human CD21 (IgG1, clone 1F8, DAKO), mouse anti-human CD35 (IgG2b, clone RLB25, Novocastra), mouse anti-human CXCL13 (IgG1, clone 53610, R&D Systems), goat anti-human CXCL13 polyclonal (IgG, AF801, R&D Systems), goat anti-human CCL21 polyclonal (IgG, AF366, R&D Systems), mouse antihuman CD31 (IgG1, clone 1A10, Novocastra) and rat antihuman/mouse PNAd (IgM, clone MECA 79, PharMingen).

Techniques: Expressing