Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: Hypoxia increased the production of S100 calcium-binding protein A8 (S100A8) in neuron and microglia and induced the release of S100A8 in SH-SY5Y cells. ( A , B ) S100A8 expression (red) were detected by immunocytochemical analysis in primary cultured neurons (NeuN, neuron marker) and cultured mixed glia (Iba1, microglial marker and GFAP, astrocyte marker) exposed to hypoxic conditions for 48 h. Scheme 25 μm. S100A8 expression was detected by western blot analysis in ( C , D ) SH-SY5Y cells and ( E , F ) BV-2 cells exposed to hypoxic conditions for 48 h. ( G , H ) S100A8 protein expression in BV-2 cells were confirmed by immunocytochemistry and ( I ) S100A8 release in SH-SY5Y was measured by enzyme-linked immunosorbent assay (ELISA) at 48 h after hypoxia. Values of * p < 0.05, ** p < 0.01, *** p < 0.001 versus control were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Binding Assay, Expressing, Cell Culture, Marker, Western Blot, Immunocytochemistry, Enzyme-linked Immunosorbent Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: S100A8 induces pro-inflammatory cytokines and inflammation in BV-2 cells. BV-2 cells were stimulated with S100A8 (10 μg/mL) for 24 h. ( A ) The supernatant was collected and TNF-α and interleukin-6 (IL-6) analyzed by ELISA. ( B ) The protein and mRNA were extracted, and the expression levels of IL-1β were assessed by ELISA and RT-qPCR. ( C – E ) The protein was extracted, separated on 10% SDS-acrylamide gels (15 μg/lane) and transferred to nitrocellulose membrane. The protein expression level was detected by western blotting with anti-ERK1/2, anti-phospho-ERK1/2 (p-ERK1/2), anti-JNK and anti-p-JNK. ( F ) Cells were pre-treated with ERK inhibitor (PD98059, 20 μM), JNK inhibitor (SP600125, 10 μM) or the equivalent volume of DMSO for 1 h, then stimulated for 24 h with LPS or S100A8 for ELISA of TNF-α, IL-6. Data from three independent experiments are presented as the means ± S.D. Values of * p < 0.05, *** p < 0.001 versus control; ### p < 0.001 versus S100A8-treated sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Membrane, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: S100A8 induces inflammasome priming by toll-like receptor (TLR)-4 receptors associated with ERK and JNK pathway in BV-2 cells. BV-2 cells were incubated for 24 h with LPS (1 μg/mL) or S100A8 (10 μg/mL) followed by Adenosine 5′-triphosphate disodium salt hydrate (ATP) (1 mM) for 1 h. ( A , B ) The NLRP3, ASC, and ( C , D ) cleaved caspase-1 were detected by western blotting. β-actin was used as an internal control. ( E , F ) BV-2 cells were lysed to whole lysates and IκB-α phosphorylation was analyzed by western blotting. ( G , H ) The translocation of nuclear factor- κB (NF-κB) was also detected by western blotting. BV-2 cells were lysed to cytosolic extracts and nucleic extracts. Lamin-B1 was used as internal controls. ( I , J ) BV-2 microglial cells were pre-treated with PD98059 (ERK inhibitor, 20 μM), SP600125 (JNK inhibitor, 10 μM), TAK-202 (TLR4 inhibitor, 10 μg/mL) or an equivalent volume of DMSO and stimulated for 24 h with LPS or S100A8. Cells harvested and lysed in RIPA buffer for western blotting of NLRP3. Results are from one experiment that is representative of at least three others. Data from three independent experiments are presented as the means ± S.D. Values of * p < 0.05, ** p < 0.01 versus control; # p < 0.05, ## p < 0.01 versus S100A8-treated sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Incubation, Western Blot, Control, Phospho-proteomics, Translocation Assay

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: S100A8 derived from neuronal cells induces NLRP3 inflammasome priming in microglia under hypoxic conditions. BV-2 cells were pre-treated with TAK-202 (TLR4 inhibitor, 10 μg/mL) for 1 h, then stimulated for 48 h in hypoxic condition with SH-SY5Y cells indirectly co-cultured in 0.4 μm pore transwell. ( A ) The protein expression level was detected by western blotting with NLRP3. β-actin was used as an internal control. ( B ) Quantitative analysis of NLRP3 levels. Data from three independent experiments are presented as the means ± S.D. Values of ** p < 0.01 versus control; # p < 0.05 versus co-cultured sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Derivative Assay, Cell Culture, Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: The expression of S100A8 in microglial cell induces apoptosis of neuronal cells in hypoxic condition. ( A , B ) SH-SY5Y cells incubated without or with S100A8 KD BV-2 cells for 48 h in hypoxic condition. Cleaved caspase-3 immunofluorescence images and were detected and quantitative analysis of the number of cleaved-caspase3-positive cells are shown in lower panel. ( C , D ) Representative Annexin-V/PI images were detected by flow cytometry. Quantitative analysis of the apoptotic rate of SH-SY5Y cells are shown in lower panel. ( E , F ) Primary neuron-glial mixed cells were transfected with S100A8 shRNA vector for 24 h followed by 48 h in hypoxic condition. Cells were harvested, and the expression protein levels of S100A8 and cleaved caspase-3 were analyzed by Western blotting. Data from three independent experiments are presented as the means ± S.D. Values of *** p < 0.001 versus control; # p < 0.05, ### p < 0.001 versus hypoxia-exposed sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Expressing, Incubation, Immunofluorescence, Flow Cytometry, Transfection, shRNA, Plasmid Preparation, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: The expression of S100A8 in microglial cell induces the Cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE 2) pathway. BV-2 cells were transfected with S100A8 shRNA or Scramble vector. After 24 h, cells were incubated in hypoxic condition for 48 h. ( A ) The mRNA and ( B ) the protein levels of S100A8 and COX-2 were detected by real-time PCR and western blotting. ( C ) Secretion of PGE 2 level analyzed by ELISA. Data from three independent experiments are presented as the means ± S.D. Values of * p < 0.05, ** p < 0.01 versus control; # p < 0.05, ### p < 0.001 versus hypoxia-exposed sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, shRNA, Plasmid Preparation, Incubation, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Mucosal immunology

Article Title: β-Glucans in food modify colonic microflora by inducing antimicrobial protein, calprotectin, in a Dectin-1-induced-IL-17F-dependent manner.

doi: 10.1038/mi.2017.86

Figure Lengend Snippet: Figure 3 Interleukin-17F (IL-17F) production is decreased in colonic lamina propria (cLP) of Clec7a / mice. (a) Cytokine gene expression in cLP was examined by quantitative real-time reverse transcription PCR (qRT-PCR), and expression levels in Clec7a / mice are indicated relative to those in wild-type (WT) mice (n ¼ 3–8/group). (b,c) IL-17F þCD45 þ cell population in cLP layer from WT or Clec7a / mice was examined by flow cytometry. (n ¼ 4–5/group). (d) S100A8 þ and IL-17F þ cells in whole colonic cells from WT mice were examined by flow cytometry. Data are representatives of two independent experiments, and in a and c are expressed as mean±s.d. *Po0.05 vs. WT control.

Article Snippet: Antibodies against mouse CD11b (M1/70), CD11c (HL3), CD45 (30-F11), Ly6G (1A8), and Ly6C (HK1.4) were obtained from BioLegend (San Diego, CA), antibodies against IL-17F (18F10) and EpCAM-1 (G8.8) were obtained from eBioscience (San Diego, CA), and antibody against S100A8 (cat. no. BAF3059) was obtained from R&D Systems.

Techniques: Gene Expression, Reverse Transcription, Quantitative RT-PCR, Expressing, Flow Cytometry, Control

Journal: Mucosal immunology

Article Title: β-Glucans in food modify colonic microflora by inducing antimicrobial protein, calprotectin, in a Dectin-1-induced-IL-17F-dependent manner.

doi: 10.1038/mi.2017.86

Figure Lengend Snippet: Figure 6 Dectin-1 ligands are not detected in intestinal commensal microbiota. (a) Wild-type (WT) mouse fecal microbiota or Candida albicans (SC5314) were pretreated with non-immune goat or rabbit serum to block nonspecific binding. Then, they were treated with PE-labeled b-glucan- recognizing protein (Pi-BGRP) or the mutant form of the b-glucan recognition site of Pi-BGRP (Pi-BGRP.M), and PE þ population was determined by flow cytometry. (b) Thioglycollate-induced peritoneal macrophages (Mfs) from WT, Clec7a / , Myd88 / , or Clec7a / Myd88 / mice were cocultured with live Alcaligenes faecalis (multiplicity of infection (MOI): 1 or 10) for 24 h and the tumor necrosis factor-a (TNF-a) concentration in the culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA; n ¼ 3/group). (c) S100A8 expression in colonic epithelial cells (cECs) from A. faecalis monocolonized (A. faecalis), germ-free (GF), or specific-pathogen-free SPF mice was examined by flow cytometry (n ¼ 3–4/group). Data in b and c are representatives of two independent experiments and are expressed as mean±s.d.

Article Snippet: Antibodies against mouse CD11b (M1/70), CD11c (HL3), CD45 (30-F11), Ly6G (1A8), and Ly6C (HK1.4) were obtained from BioLegend (San Diego, CA), antibodies against IL-17F (18F10) and EpCAM-1 (G8.8) were obtained from eBioscience (San Diego, CA), and antibody against S100A8 (cat. no. BAF3059) was obtained from R&D Systems.

Techniques: Blocking Assay, Binding Assay, Labeling, Mutagenesis, Flow Cytometry, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: E-Selectin-Dependent Inflammation and Lipolysis in Adipose Tissue Exacerbate Steatosis-to-NASH Progression via S100A8/9

doi: 10.1016/j.jcmgh.2021.08.002

Figure Lengend Snippet: Antibodies

Article Snippet: S100A8/A9 (mouse) , 1:500 (staining) 1:5000 (WB) , Novus Biologicals.

Techniques: Staining

Journal: Biomolecules

Article Title: Evaluation of Plasma-Derived hsa_circ_003077 for Non-Invasive Diagnosis of Alzheimer’s Disease

doi: 10.3390/biom16030356

Figure Lengend Snippet: Plasma glial and receptor biomarkers in Alzheimer’s disease (AD) patients and cognitively healthy controls (HC). ( a ) GFAP, ( b ) NfL, ( c ) Tyro3, and ( d ) AXL levels were measured in plasma. Data are presented as mean ± SEM. Statistical comparisons were performed using the Mann–Whitney U test. Statistical significance is indicated as ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: The Human Tyro3 DuoSet ELISA kit (Catalog No: DY8596-05) supplied by R&D Systems (Minneapolis, MN, USA) was used to measure Tyro3 levels, while the Human AXL DuoSet ELISA kit (Catalog No: DAXL00; R&D Systems, Minneapolis, MN, USA) was used to measure AXL levels.

Techniques: Clinical Proteomics, MANN-WHITNEY

Journal: Biomolecules

Article Title: Evaluation of Plasma-Derived hsa_circ_003077 for Non-Invasive Diagnosis of Alzheimer’s Disease

doi: 10.3390/biom16030356

Figure Lengend Snippet: Diagnostic performance of plasma biomarkers for differentiating Alzheimer’s disease (AD) from cognitively healthy controls (HC) based on ROC curve analysis. Receiver operating characteristic (ROC) curves were generated to assess the diagnostic accuracy of plasma biomarkers. The area under the ROC curve (AUC) was used to evaluate biomarker performance: 0.90–1.00, superior diagnostic efficacy; 0.80–0.89, good diagnostic efficacy; 0.70–0.79, moderate diagnostic efficacy; <0.70, poor/substandard diagnostic efficacy. Data are presented as mean ± SEM. Statistical comparisons were performed using the Mann–Whitney U test. hsa_circ_003077 exhibited the highest diagnostic accuracy (AUC = 0.90; 95% CI: 0.82–0.97), followed by NfL (AUC = 0.75) and pTau-217 (AUC = 0.77). AXL and GFAP showed lower diagnostic performance (AUC = 0.63 and 0.69, respectively), while pTau-181 and Tyro3 demonstrated substandard efficacy (AUC = 0.63 each).

Article Snippet: The Human Tyro3 DuoSet ELISA kit (Catalog No: DY8596-05) supplied by R&D Systems (Minneapolis, MN, USA) was used to measure Tyro3 levels, while the Human AXL DuoSet ELISA kit (Catalog No: DAXL00; R&D Systems, Minneapolis, MN, USA) was used to measure AXL levels.

Techniques: Diagnostic Assay, Clinical Proteomics, Generated, Biomarker Discovery, MANN-WHITNEY

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Elevated Levels of Neutrophil Activated Proteins, Alpha-Defensins (DEFA1), Calprotectin (S100A8/A9) and Myeloperoxidase (MPO) Are Associated With Disease Severity in COVID-19 Patients

doi: 10.3389/fcimb.2021.751232

Figure Lengend Snippet: Details of the COVID-19 patients examined at presentation.

Article Snippet: Levels of alpha defensins, DEFA1 and S100A8/A9 in serum and plasma were quantified using commercially available ELISA kits, Neutrophil DEFA1 (USCN, Wuhan) & quantikine ELISA for human S100A8/A9 Heterodimer (R & D systems, Minneapolis) as per manufacturer’s protocols.

Techniques:

Journal: Acta dermato-venereologica

Article Title: Crisaborole Inhibits Itch and Pain by Preventing Neutrophil Infiltration in a Mouse Model of Atopic Dermatitis.

doi: 10.2340/actadv.v103.13382

Figure Lengend Snippet: Fig. 2. Effects of crisaborole on neutrophil infiltration in atopic dermatitis mouse model. (A–F) Skin was dissected from mice on day 8 after EtOH (A, E, F), MC903 (MC: B, E, F), MC903+vehicle (MC+VH: C, E, F), or MC903+crisaborole treatment (MC+Cris: D, E, F). Skin sections were immunostained with an antibody for PGP9.5 and Ly6G to visualize nerve fibres (green) and neutrophils (red), respectively. Dotted lines indicate the dermal-epidermal junction. White arrows indicate neutrophils contact with nerve fibres. (E) Scale bar indicates 20 µm. Total number of neutrophils in the epidermis was quantified in EtOH (white bars), MC (green bars), MC+VH (dark grey bars), and MC+Cris mice (blue bars). Error bars are SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001, significant difference (1-way analysis of variance (ANOVA) followed by Tukey test, n = 5/group). (F) As in (E) for epidermal neutrophils in contact with nerves. (G and H) Skin sections were immunostained with an antibody for Ly6G and S100A8 or S100A9.

Article Snippet: Skin sections were incubated with 5% goat or donkey serum and 0.2% Triton X-100 in PBS, and then immunostained with a primary antibody directed against PGP 9.5 (1:2000; Chemicon (Millipore), Billerica, MA, USA; Cat# AB1761-I, RRID:AB_2868444), to label all epidermal nerve fibres at 4°C overnight, followed by incubation with the corresponding secondary antibody conjugated with AlexaFluor 488 (1:300; Life Technologies Inc., Grand Island, NY, USA) for 2 h. Subsequently, the sections were immunostained with rat Ly6G antibody (1:500; Bio X Cell, Lebanon, NH, USA; Cat# BE0075-1, RRID:AB_1107721), the most commonly used marker for neutrophils at 4°C overnight, followed by incubation with the corresponding secondary antibody conjugated with AlexaFluor 633 (1:300; Life Technologies Inc.) for 2 h. For S100A8 or S100A9 experiments, the sections were incubated with goat S100A9 antibody (1:400; R&D systems, Minneapolis, MN, USA; Cat# AF2065, RRID:AB_2184263) or rat S100A8 antibody (1:50; R&D Systems; Cat# MAB3059, RRID:AB_2184252) followed by goat secondary antibody conjugated with AlexaFluor 555 (1:300; Life Technologies Inc.) or rat secondary antibody conjugated with AlexaFluor 633 (1:300; Life Technologies Inc.) for 2 h, respectively.

Techniques:

Journal: Acta dermato-venereologica

Article Title: Crisaborole Inhibits Itch and Pain by Preventing Neutrophil Infiltration in a Mouse Model of Atopic Dermatitis.

doi: 10.2340/actadv.v103.13382

Figure Lengend Snippet: Fig. 3. S100A8/A9 facilitates pruritogen- or algogen-evoked scratching and wiping behaviours in mice. (A) S100A8/A9 (1, 10, 100 ng) or vehicle (phosphate-buffered saline; PBS) was intradermally injected into the nape of the neck. Following the injection, scratch bouts were counted over a 30-min period. Error bars are standard error of mean (SEM) n = 6–8. (B, C) S100A8/A9 (1, 10 ng) or vehicle was intradermally injected into the nape of the neck with either histamine (54 nmol: B) or chloroquine (97 nmol: C). Error bars are SEM n = 6–8. *p < 0.05, **p < 0.01, ****p < 0.0001, significant difference from the vehicle-treated group. One-way analysis of variance (ANOVA) followed by the post hoc Tukey test. F (2, 19)=18.60, p < 0.0001 for histamine. F (2, 15)=6.353, p = 0.01 for chloroquine. (D–I) S100A8/A9 (10 ng) or vehicle was intradermally injected into the cheek with either histamine (54 nmol: D, G), chloroquine (97 nmol: E, H), or capsaicin (33 nmol: F, I). Error bars are SEM n = 5–6. *p < 0.05, ***p < 0.001, ****p < 0.0001, significant difference from the vehicle-treated group. Two-tailed unpaired t-test.

Article Snippet: Skin sections were incubated with 5% goat or donkey serum and 0.2% Triton X-100 in PBS, and then immunostained with a primary antibody directed against PGP 9.5 (1:2000; Chemicon (Millipore), Billerica, MA, USA; Cat# AB1761-I, RRID:AB_2868444), to label all epidermal nerve fibres at 4°C overnight, followed by incubation with the corresponding secondary antibody conjugated with AlexaFluor 488 (1:300; Life Technologies Inc., Grand Island, NY, USA) for 2 h. Subsequently, the sections were immunostained with rat Ly6G antibody (1:500; Bio X Cell, Lebanon, NH, USA; Cat# BE0075-1, RRID:AB_1107721), the most commonly used marker for neutrophils at 4°C overnight, followed by incubation with the corresponding secondary antibody conjugated with AlexaFluor 633 (1:300; Life Technologies Inc.) for 2 h. For S100A8 or S100A9 experiments, the sections were incubated with goat S100A9 antibody (1:400; R&D systems, Minneapolis, MN, USA; Cat# AF2065, RRID:AB_2184263) or rat S100A8 antibody (1:50; R&D Systems; Cat# MAB3059, RRID:AB_2184252) followed by goat secondary antibody conjugated with AlexaFluor 555 (1:300; Life Technologies Inc.) or rat secondary antibody conjugated with AlexaFluor 633 (1:300; Life Technologies Inc.) for 2 h, respectively.

Techniques: Saline, Injection, Two Tailed Test

Journal: Acta dermato-venereologica

Article Title: Crisaborole Inhibits Itch and Pain by Preventing Neutrophil Infiltration in a Mouse Model of Atopic Dermatitis.

doi: 10.2340/actadv.v103.13382

Figure Lengend Snippet: Fig. 4. Effects of S100A8/A9 on pruritogen- or algogen-induced calcium response in mouse dorsal root ganglion (DRG) neurones. (A) Time-course graph of histamine-induced calcium response in DRG neurones. DRG neurones were treated with vehicle (black line) or S100A8/A9 (blue line) with a subsequent challenge with histamine (100 μM). (B) As in (A) for chloroquine (100 μM). (C) As in (A) for capsaicin (1 μM). (D) Averaged area under the curve of the calcium responses to histamine. DRG neurones were treated with vehicle (black column) or S100A8/A9 (blue column). Error bars are SEM n = 19–27. ***p < 0.001, a significant difference from the vehicle group. 2-tailed unpaired t-test. (E) As in (D) for chloroquine. n = 11–22. (F) As in (D) for capsaicin. n = 13–14. (G) Proportions of histamine-, chloroquine-, or capsaicin-responsive DRG neurones pre-treated with vehicle (black column) or S100A8/A9 (blue column). *p < 0.05. Fisher exact test. n = 69–128.

Article Snippet: Skin sections were incubated with 5% goat or donkey serum and 0.2% Triton X-100 in PBS, and then immunostained with a primary antibody directed against PGP 9.5 (1:2000; Chemicon (Millipore), Billerica, MA, USA; Cat# AB1761-I, RRID:AB_2868444), to label all epidermal nerve fibres at 4°C overnight, followed by incubation with the corresponding secondary antibody conjugated with AlexaFluor 488 (1:300; Life Technologies Inc., Grand Island, NY, USA) for 2 h. Subsequently, the sections were immunostained with rat Ly6G antibody (1:500; Bio X Cell, Lebanon, NH, USA; Cat# BE0075-1, RRID:AB_1107721), the most commonly used marker for neutrophils at 4°C overnight, followed by incubation with the corresponding secondary antibody conjugated with AlexaFluor 633 (1:300; Life Technologies Inc.) for 2 h. For S100A8 or S100A9 experiments, the sections were incubated with goat S100A9 antibody (1:400; R&D systems, Minneapolis, MN, USA; Cat# AF2065, RRID:AB_2184263) or rat S100A8 antibody (1:50; R&D Systems; Cat# MAB3059, RRID:AB_2184252) followed by goat secondary antibody conjugated with AlexaFluor 555 (1:300; Life Technologies Inc.) or rat secondary antibody conjugated with AlexaFluor 633 (1:300; Life Technologies Inc.) for 2 h, respectively.

Techniques: