rabbit anti mouse tnfr1  (Danaher Inc)

Journal: Arthritis Research & Therapy

Article Title: Induction of tumour necrosis factor receptor-expressing macrophages by interleukin-10 and macrophage colony-stimulating factor in rheumatoid arthritis

doi: 10.1186/ar2015

Figure Lengend Snippet: Expression of type 1 and type 2 tumour necrosis factor receptor (TNFR1/2) and macrophage colony-stimulating factor receptor (M-CSFR) on monocytes of patients with rheumatoid arthritis (RA) and healthy controls (HCs). Peripheral blood mononuclear cells were stained with immunoglobulin (Ig) G 2a anti-TNFR1 antibody, rabbit IgG anti-M-CSFR polyclonal antibody, or isotype-matched control antibody, followed by fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG 2a antibody or phycoerythrin-conjugated anti-rabbit IgG antibody, and with FITC-conjugated anti-TNFR2 antibody. TNFR1, TNFR2, and M-CSFR expression on monocytes from patients with RA and HCs was analysed by flow cytometric analysis. The intensity of cytokine receptor was expressed by the ratio of the mean fluorescence intensity (MFI) of staining with anti-cytokine receptor antibody to the MFI of control antibody. Values are the mean ± standard error of the mean. n, number of samples tested.

Article Snippet: Double immunofluorescence was performed by serially incubating sections with 10 μg/ml of mouse IgG 1 anti-IL-10R1 mAb, rabbit IgG anti-TNFR1 Ab, rabbit IgG anti-TNFR2 Ab (Santa Cruz Biotechnology, Inc.), rabbit IgG anti-M-CSFR Ab, mouse IgG 1 anti-CD68 mAb (BD Biosciences), and isotype-matched control Abs at 4°C overnight, followed by incubation with rhodamine-conjugated goat anti-mouse IgG 1 mAb (Santa Cruz Biotechnology, Inc.) and FITC-conjugated anti-rabbit IgG mAb (Santa Cruz Biotechnology, Inc.) for 30 minutes at room temperature.

Techniques: Expressing, Staining, Fluorescence

Journal: Arthritis Research & Therapy

Article Title: Induction of tumour necrosis factor receptor-expressing macrophages by interleukin-10 and macrophage colony-stimulating factor in rheumatoid arthritis

doi: 10.1186/ar2015

Figure Lengend Snippet: Induction of mRNA expression of type 1 and type 2 tumour necrosis factor receptor (TNFR1/2) in monocytes by interleukin-10 (IL-10). Adhered monocytes from patients with rheumatoid arthritis (RA) and healthy controls (HCs) (5 × 10 5 cells in culture medium with 10% fetal calf serum) were incubated for 12 hours with or without 25 ng/ml IL-10. Total cellular RNA was extracted from these monocytes, and mRNA expression was analysed by real-time polymerase chain reaction analysis as described in Materials and methods. Levels of TNFR1 and TNFR2 mRNAs were normalised relative to β-actin expression, and the ratio of the levels of monocytes incubated with IL-10 to the levels without IL-10 was determined. Values are the mean ± standard error of the mean. n, number of samples tested.

Article Snippet: Double immunofluorescence was performed by serially incubating sections with 10 μg/ml of mouse IgG 1 anti-IL-10R1 mAb, rabbit IgG anti-TNFR1 Ab, rabbit IgG anti-TNFR2 Ab (Santa Cruz Biotechnology, Inc.), rabbit IgG anti-M-CSFR Ab, mouse IgG 1 anti-CD68 mAb (BD Biosciences), and isotype-matched control Abs at 4°C overnight, followed by incubation with rhodamine-conjugated goat anti-mouse IgG 1 mAb (Santa Cruz Biotechnology, Inc.) and FITC-conjugated anti-rabbit IgG mAb (Santa Cruz Biotechnology, Inc.) for 30 minutes at room temperature.

Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction

Journal: Arthritis Research & Therapy

Article Title: Induction of tumour necrosis factor receptor-expressing macrophages by interleukin-10 and macrophage colony-stimulating factor in rheumatoid arthritis

doi: 10.1186/ar2015

Figure Lengend Snippet: Cytokine receptor induction on monocytes stimulated with interleukin-10 (IL-10) and macrophage colony-stimulating factor (M-CSF). (a) Induction of monocyte expression of M-CSF receptor (M-CSFR) by IL-10 and/or M-CSF. Purified normal monocytes from different individuals (5 × 10 5 cells in culture medium with 10% fetal calf serum) were incubated for 3 days with or without 50 ng/ml M-CSF, 25 ng/ml IL-10, or IL-10 plus M-CSF. M-CSFR expression was analysed by flow cytometric analysis, and the cytokine-mediated M-CSFR induction was expressed by the ratio of the mean fluorescence intensity (MFI) of M-CSFR expression with cytokines to the MFI without cytokines. Values are the mean ± standard error of the mean. (b) Representative histographic patterns of type 1 and type 2 tumour necrosis factor receptor (TNFR1/2) expression on monocytes incubated with or without IL-10 plus M-CSF. TNFR1 and TNFR2 expression was analysed by flow cytometric analysis. (c) Induction of monocyte expression of TNFR1 and TNFR2 by IL-10 and/or M-CSF. The cytokine-mediated TNFR1/2 induction was expressed by the ratio of the mean fluorescence intensity (MFI) of TNFR1/2 expression with cytokines to the MFI without cytokines. n, number of samples tested.

Article Snippet: Double immunofluorescence was performed by serially incubating sections with 10 μg/ml of mouse IgG 1 anti-IL-10R1 mAb, rabbit IgG anti-TNFR1 Ab, rabbit IgG anti-TNFR2 Ab (Santa Cruz Biotechnology, Inc.), rabbit IgG anti-M-CSFR Ab, mouse IgG 1 anti-CD68 mAb (BD Biosciences), and isotype-matched control Abs at 4°C overnight, followed by incubation with rhodamine-conjugated goat anti-mouse IgG 1 mAb (Santa Cruz Biotechnology, Inc.) and FITC-conjugated anti-rabbit IgG mAb (Santa Cruz Biotechnology, Inc.) for 30 minutes at room temperature.

Techniques: Expressing, Purification, Incubation, Fluorescence

Journal: Arthritis Research & Therapy

Article Title: Induction of tumour necrosis factor receptor-expressing macrophages by interleukin-10 and macrophage colony-stimulating factor in rheumatoid arthritis

doi: 10.1186/ar2015

Figure Lengend Snippet: Expression of type 1 interleukin-10 receptor (IL-10R1), macrophage colony-stimulating factor receptor (M-CSFR), and type 1 and 2 tumour necrosis factor receptor (TNFR1/2) in synovial tissue (ST) from patients with rheumatoid arthritis. ST sections were stained with mouse immunoglobulin (Ig) G 1 anti-IL-10R1 monoclonal antibody (mAb), rabbit IgG anti-TNFR1 Ab, rabbit IgG anti-TNFR2 Ab, rabbit IgG anti-M-CSFR Ab, mouse IgG 1 anti-CD68 mAb, and isotype-matched control Abs, followed by incubation with rhodamine-conjugated goat anti-mouse IgG 1 mAb and fluorescein isothiocyanate-conjugated anti-rabbit IgG mAb. Two-color immunofluorescence confocal images were obtained for expression of IL-10R1 (red staining), M-CSFR (red), TNFR1 (green), TNFR2 (green), and CD68 (green). The two images were superimposed, and double-positive cells are shown in yellow. Similar staining patterns were obtained in additional analyses from five ST samples from different patients.

Article Snippet: Double immunofluorescence was performed by serially incubating sections with 10 μg/ml of mouse IgG 1 anti-IL-10R1 mAb, rabbit IgG anti-TNFR1 Ab, rabbit IgG anti-TNFR2 Ab (Santa Cruz Biotechnology, Inc.), rabbit IgG anti-M-CSFR Ab, mouse IgG 1 anti-CD68 mAb (BD Biosciences), and isotype-matched control Abs at 4°C overnight, followed by incubation with rhodamine-conjugated goat anti-mouse IgG 1 mAb (Santa Cruz Biotechnology, Inc.) and FITC-conjugated anti-rabbit IgG mAb (Santa Cruz Biotechnology, Inc.) for 30 minutes at room temperature.

Techniques: Expressing, Staining, Incubation, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: TRAPS mutations in Tnfrsf1a decrease the responsiveness to TNFα via reduced cell surface expression of TNFR1

doi: 10.3389/fimmu.2022.926175

Figure Lengend Snippet: TRAPS mutations strongly suppressed lethal response by LPS and D-galactosamine and diminished TNFα-mediated arthritis. (A-C) T79M and G87V TRAPS mutant mice and TNFR1 KO mice were intraperitoneally administrated with LPS (100 μg/kg body weight) and D-galactosamine (400 mg/kg body weight). Survival rates of the mice after the administration of LPS and D-galactosamine. Survival rates of WT ( n = 23), T79M strain (A) ; Het ( n =18), Hom ( n = 9), G87V strain (B) ; Het ( n =13), Hom ( n = 13), and TNFR1 KO strain (C) ; Het ( n =11), Hom ( n = 9). Gray, orange, and blue lines indicate WT, heterozygotes, and homozygotes, respectively. p -values for the differences between subgroups of mice were calculated by log-rank test using stratified analysis. (D, E) T79M or G87V mutant mice were crossed with TNFtg mice, and the severity of arthritis was evaluated. WT ( n = 5), T79M Het ( n = 4), T79M Hom ( n = 3), G87V Het ( n = 3), G87V Hom ( n = 3), TNFtg ( n = 5), TNFtg/T79M Het ( n = 4), TNFtg/T79M Hom ( n = 3), TNFtg/G87V Het ( n = 4), TNFtg/G87V Hom ( n = 2). (D) Representative images of the hind paw of TNFtg T79M heterozygous mouse and TNFtg mouse. (E) Arthritis scores of the mice at the age of 17 weeks. ** p <0.01 vs . any other groups. WT, wild-type; TNFtg, TNFα transgenic; TNFα, Tumor necrosis factor α; TRAPS, TNF receptor-associated periodic syndrome; IL, interleukin; LPS, lipopolysaccharide; TNFR1, TNF receptor type I; TNFtg, Human TNFα-transgenic mice; Het, heterozygote; Hom, homozygote. * p <0.01; n.s., not significant.

Article Snippet: The following primary antibodies were used for western blot analysis: anti-mouse TNFR1 (13377), p38 (8690), extracellular-signal-regulated kinase (ERK) (4695), jun N-terminal kinase (JNK) (9252), NF-κB p65 (8242), phospho-p38 Thr180/Tyr182 (4511), phospho-ERK Thr202/Tyr204 (4370), phospho-NF-κB p65 Ser536 (3033), ATF-6 (65880), IRE1α (3294; all from Cell Signaling Technology, Danvers, MA, USA), TNFR1 (AF-425-PB, R&D Systems, Minneapolis, MN, USA), TNFR2 (ab109322, Abcam, Cambridge, UK), and actin (A2066, Sigma-Aldrich).

Techniques: Mutagenesis, Transgenic Assay

Journal: Frontiers in Immunology

Article Title: TRAPS mutations in Tnfrsf1a decrease the responsiveness to TNFα via reduced cell surface expression of TNFR1

doi: 10.3389/fimmu.2022.926175

Figure Lengend Snippet: No substantial changes in the responsiveness to LPS in the TRAPS mutant macrophages. Bone marrow-derived macrophages were stimulated with LPS (100 ng/mL). After stimulation, the RNA and supernatant samples were collected at the indicated time points. Cell culture experiments were performed separately for each strain. (A) qPCR analysis. The mRNA expression levels of Tnf and Il1b were determined. Gray, orange, blue, and dark gray lines indicate WT, heterozygous, homozygous, and TNFR1KO mice, respectively. (B) Relative protein levels of TNFα and IL-1β in the culture supernatants. Culture supernatants were collected 6 h after LPS stimulation, and the concentrations of TNFα and IL-1β were determined by ELISA. Levels were calculated relative to those of the WT in each strain. TNFα, Tumor necrosis factor α; TRAPS, TNF receptor-associated periodic syndrome; IL, interleukin; LPS, lipopolysaccharide; TNFR1, TNF receptor type I; KO, knockout; qPCR, real-time quantitative polymerase chain reaction; WT, wild-type; Het, heterozygote; Hom, homozygote.

Article Snippet: The following primary antibodies were used for western blot analysis: anti-mouse TNFR1 (13377), p38 (8690), extracellular-signal-regulated kinase (ERK) (4695), jun N-terminal kinase (JNK) (9252), NF-κB p65 (8242), phospho-p38 Thr180/Tyr182 (4511), phospho-ERK Thr202/Tyr204 (4370), phospho-NF-κB p65 Ser536 (3033), ATF-6 (65880), IRE1α (3294; all from Cell Signaling Technology, Danvers, MA, USA), TNFR1 (AF-425-PB, R&D Systems, Minneapolis, MN, USA), TNFR2 (ab109322, Abcam, Cambridge, UK), and actin (A2066, Sigma-Aldrich).

Techniques: Mutagenesis, Derivative Assay, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Knock-Out, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: TRAPS mutations in Tnfrsf1a decrease the responsiveness to TNFα via reduced cell surface expression of TNFR1

doi: 10.3389/fimmu.2022.926175

Figure Lengend Snippet: Decreased responsiveness to TNFα in the TRAPS mutations. Primary murine bone marrow-derived macrophages were stimulated with 100 ng/mL TNFα. RNA samples were collected at indicated time points. Cell culture experiments were performed separately for each strain. The mRNA expression levels of Tnf and Il1b were determined using qPCR. The WT levels at 0 h were set to 1. Gray, orange, blue, and dark gray lines indicate WT, heterozygous, homozygous, and TNFR1KO mice, respectively. TNFα, Tumor necrosis factor α; TRAPS, TNF receptor-associated periodic syndrome; WT, wild-type; Het, heterozygote; Hom, homozygote.

Article Snippet: The following primary antibodies were used for western blot analysis: anti-mouse TNFR1 (13377), p38 (8690), extracellular-signal-regulated kinase (ERK) (4695), jun N-terminal kinase (JNK) (9252), NF-κB p65 (8242), phospho-p38 Thr180/Tyr182 (4511), phospho-ERK Thr202/Tyr204 (4370), phospho-NF-κB p65 Ser536 (3033), ATF-6 (65880), IRE1α (3294; all from Cell Signaling Technology, Danvers, MA, USA), TNFR1 (AF-425-PB, R&D Systems, Minneapolis, MN, USA), TNFR2 (ab109322, Abcam, Cambridge, UK), and actin (A2066, Sigma-Aldrich).

Techniques: Derivative Assay, Cell Culture, Expressing

Journal: Frontiers in Immunology

Article Title: TRAPS mutations in Tnfrsf1a decrease the responsiveness to TNFα via reduced cell surface expression of TNFR1

doi: 10.3389/fimmu.2022.926175

Figure Lengend Snippet: Decreased cell surface expression of TNFR1 in the TRAPS mutant cells. The expression of TNFR1 and TNFR2 were determined in primary murine bone marrow-derived macrophages and peritoneal macrophages. (A) mRNA expression levels of Tnfrsf1a and Tnfrsf1b in bone marrow-derived macrophages. (B) Immunoblot analysis of TNFR1 and TNFR2 expression in the bone marrow-derived macrophages. Two different antibodies against TNFR1 were used: Ab#1 (13377, Cell Signaling Technology) recognizes aa 29–43 (extracellular domain), and Ab#2 (AF-425-PB, R&D Systems) recognizes the C-terminal intracellular region. (C–E) Flow cytometric analysis of TNFR1 and TNFR2 expression. Peritoneal exudate cells were collected from the indicated mice 3 days after the intraperitoneal administration of thioglycolate. (C) Representative histograms of the cell surface expression of TNFR1 and TNFR2. The expression levels of TNFR1 (D) and TNFR2 (E) on the surface of CD11b-positive cells were determined by flow cytometry. The cells within the horizontal line on each histogram were recognized as TNFR1- or TNFR2-positive cells. The proportions of the positive cells were calculated relative to those of the WT in each strain. The following antibodies were used: TNFR1 (113005, BioLegend) and TNFR2 (113405, BioLegend). Both antibodies recognize the extracellular domains. (F, G) ELISA for soluble TNFR1 (sTNFR1) in the culture supernatant of bone marrow-derived macrophages. Culture supernatant was collected before (F) and 24 h after (G) LPS stimulation. Concentrations of sTNFR1 in the culture supernatant were measured by ELISA, and the levels were calculated relative to that of the WT in each strain. TRAPS, TNF receptor-associated periodic syndrome; LPS, lipopolysaccharide; ELISA, enzyme-linked immunosorbent assay; WT, wild-type; Het, heterozygote; Hom, homozygote.

Article Snippet: The following primary antibodies were used for western blot analysis: anti-mouse TNFR1 (13377), p38 (8690), extracellular-signal-regulated kinase (ERK) (4695), jun N-terminal kinase (JNK) (9252), NF-κB p65 (8242), phospho-p38 Thr180/Tyr182 (4511), phospho-ERK Thr202/Tyr204 (4370), phospho-NF-κB p65 Ser536 (3033), ATF-6 (65880), IRE1α (3294; all from Cell Signaling Technology, Danvers, MA, USA), TNFR1 (AF-425-PB, R&D Systems, Minneapolis, MN, USA), TNFR2 (ab109322, Abcam, Cambridge, UK), and actin (A2066, Sigma-Aldrich).

Techniques: Expressing, Mutagenesis, Derivative Assay, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Acta Neuropathologica Communications

Article Title: Characterization of the TNF and IL-1 systems in human brain and blood after ischemic stroke

doi: 10.1186/s40478-020-00957-y

Figure Lengend Snippet: Characterization of cellular localization and plasma levels of TNF, TNFR1, and TNFR2 in ischemic stroke. a TNF immunohistochemical staining showing a TNF + neuron (arrowhead) in a ≤ 2-day-old infarct. b TNF + glial- and macrophage-like cells (arrows) located in a 3–7-day-old infarct. c TNF + cells (arrows) and TNF + macrophages extravasating from capillaries (arrows in insert) in a ≥ 8-day-old infarct. d Scoring of TNF staining intensity showing significantly higher TNF expression in neurons located in I/PI in ≤2-day-old infarcts compared to ≥8-day-old infarcts and in neurons located in NAT in ≤2-day-old infarcts compared to both 3–7-day-old and ≥ 8-day-old infarcts (upper graph). TNF expression was comparable in glial cells located in I/PI and NAT in ≤2-day-old and 3–7-day-old infarcts, with significantly increased expression in I/PI in ≥8-day-old infarcts compared to ≤2-day- and 3–7-day-old infarcts and compared to NAT (middle graph). At all timepoints, TNF expression in macrophages was only observed in I/PI (lower graph). e TNFR1 immunohistochemical staining demonstrating a TNFR1 + glial cell (arrow) and TNFR1 + neuron (arrowhead) in a ≤ 2-day-old infarct. f TNFR1 + neurons (arrowheads) in a 3–7-day-old infarct. g TNFR1 + neuron (arrowhead) in a ≥ 8-day-old infarct. h Scoring of staining intensity showed that TNFR1 expression was comparable in neurons and glia in I/PI and NAT at all timepoints. TNFR1 was mainly expressed in macrophages located in I/PI and absent from NAT. i TNFR2 immunohistochemical staining demonstrating TNFR2 + glial end-feet encircling the blood vessels (arrow) and a TNFR2 + glia cell (insert) in ≤2-day-old infarcts. j TNFR2 + cell (arrow) in a 3–7-day-old infarct. k TNFR2 + glia in a ≥ 8-day-old infarct. l Scoring of TNFR2 staining intensity showing comparable TNFR2 expression in glia located in I/PI and NAT in ≤2-day-old and 3–7-day-old infarcts and with a significant increase in TNFR2 expression in glia located in I/PI compared to NAT in ≥8-day-old infarcts. TNFR2 expression was only found in macrophages located in I/PI. No neurons were found to express TNFR2. m Immunofluorescent staining demonstrating co-localization of TNF (red) with NeuN + (green) neurons (arrows). n Immunofluorescent staining demonstrating co-localization of TNFR1 (red) with NF + (green) neurons and their proximal dentrites (arrows). o Immunofluorescent staining demonstrating co-localization of TNFR2 (green) with GFAP + astrocytes (red). p High magnification of ( o ). q V-Plex analysis demonstrating comparable plasma TNF levels between controls and ischemic stroke patients at < 8 and 72 h after symptom onset. r-s V-Plex analysis demonstrating significantly increased plasma TNFR1 (r) and TNFR2 (s) levels in ischemic stroke patients < 8 h after symptom onset compared to controls. For TNF, CV > 25% in three control samples and one < 8-h sample. For TNFR1, CV > 25% in four < 8-h samples, 12 control samples, and one 72-h sample. For TNFR2, CV > 25% in three < 8-h samples, 11 control samples, and three 72-h samples. GFAP, glial fibrillary acidic protein; I/PI, infarct/peri-infarct; NF, neurofilament; NAT, normal appearing tissue; TNF, tumor necrosis factor; TNFR1, tumor necrosis factor receptor 1; TNFR2, tumor necrosis factor receptor 2. Results are presented as mean ± SD (staining intensity) or mean with IQR (V-plex analysis). * p < 0.05, ** p < 0.01, *** p < 0.001; one-way ANOVA followed by Sidak’s multiple comparisons test (scoring of staining intensity, n = 3–9/group) or Kruskal Wallis test followed by Dunn’s multiple comparison test (V-plex analysis). Scale bars: a-c, e-g, i-k, p, inserts = 40 μm, and m-o = 100 μm

Article Snippet: Sections were stained using the following primary antibodies: mouse anti-CD68 (clone PG-M1, 1:100, Dako), mouse anti-CD45 (clone 2B11, 1:200, Dako), rabbit anti-Iba1 (ionized calcium binding adaptor molecule 1, 1:1000, Wako), rabbit anti-GFAP (1:2000, Dako), mouse anti-neurofilament (NF) (phosphorylated and non-phosphorylated NF-heavy chain; clone N52, 1:1000, Sigma-Aldrich), mouse anti-IL-1α (clone 4414, 1:1200, R&D Systems), mouse anti-IL-1β (clone 2E8, 1:50, BioRad), rabbit anti-TNF (1:100, ThermoFisher Scientific), rabbit anti-TNFR1 (clone H-271, 1:50, Santa Cruz), rabbit anti-TNFR2 (1:50, Sigma-Aldrich), and rat anti-IL-1Ra (clone 40,007, 1:1500, R&D Systems).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Acta Neuropathologica Communications

Article Title: Characterization of the TNF and IL-1 systems in human brain and blood after ischemic stroke

doi: 10.1186/s40478-020-00957-y

Figure Lengend Snippet: Characterization of glial markers, IL-1, TNF, and TNF receptors in post-mortem tissue sections. a-j Immunohistochemical staining of parallel tissue sections from a < 1-day-old infarct for glial markers Iba1 ( a ), CD45 ( b ), and CD68 ( c ), and for cytokines IL-1β ( d ), IL-1α ( e ), IL-1Ra ( f ), and TNF ( g ), and for TNFR1 ( h ), TNFR2 ( i ), and the astrocyte marker GFAP ( j ). GFAP, glial fibrillary acidic protein; IL-1α, interleukin-1alpha; IL-1β, interleukin-1beta; IL-1Ra, interleukin-1 receptor antagonist; TNF, tumor necrosis factor; TNFR1, tumor necrosis factor receptor 1; TNFR2, tumor necrosis factor receptor 2. Scale bar = 40 μm

Article Snippet: Sections were stained using the following primary antibodies: mouse anti-CD68 (clone PG-M1, 1:100, Dako), mouse anti-CD45 (clone 2B11, 1:200, Dako), rabbit anti-Iba1 (ionized calcium binding adaptor molecule 1, 1:1000, Wako), rabbit anti-GFAP (1:2000, Dako), mouse anti-neurofilament (NF) (phosphorylated and non-phosphorylated NF-heavy chain; clone N52, 1:1000, Sigma-Aldrich), mouse anti-IL-1α (clone 4414, 1:1200, R&D Systems), mouse anti-IL-1β (clone 2E8, 1:50, BioRad), rabbit anti-TNF (1:100, ThermoFisher Scientific), rabbit anti-TNFR1 (clone H-271, 1:50, Santa Cruz), rabbit anti-TNFR2 (1:50, Sigma-Aldrich), and rat anti-IL-1Ra (clone 40,007, 1:1500, R&D Systems).

Techniques: Immunohistochemical staining, Staining, Marker

Journal: Acta Neuropathologica Communications

Article Title: Characterization of the TNF and IL-1 systems in human brain and blood after ischemic stroke

doi: 10.1186/s40478-020-00957-y

Figure Lengend Snippet: Characterization of IL-1, TNF, and TNF receptors TNFR1, and TNFR2 in post-mortem tissue sections. a-f Immunohistochemical staining of parallel tissue sections from a > 7-day-old infarct for cytokines IL-1β (arrows in a ), IL-1α (arrows in b ), IL-1Ra (arrows in c ), and TNF (arrows in d ), and for TNFR1 (arrows in e ), and TNFR2 (arrows in f ). IL-1α, interleukin-1alpha; IL-1β, interleukin-1beta; IL-1Ra, interleukin-1 receptor antagonist; TNF, tumor necrosis factor; TNFR1, tumor necrosis factor receptor 1; TNFR2, tumor necrosis factor receptor 2. Scale bar = 40 μm

Article Snippet: Sections were stained using the following primary antibodies: mouse anti-CD68 (clone PG-M1, 1:100, Dako), mouse anti-CD45 (clone 2B11, 1:200, Dako), rabbit anti-Iba1 (ionized calcium binding adaptor molecule 1, 1:1000, Wako), rabbit anti-GFAP (1:2000, Dako), mouse anti-neurofilament (NF) (phosphorylated and non-phosphorylated NF-heavy chain; clone N52, 1:1000, Sigma-Aldrich), mouse anti-IL-1α (clone 4414, 1:1200, R&D Systems), mouse anti-IL-1β (clone 2E8, 1:50, BioRad), rabbit anti-TNF (1:100, ThermoFisher Scientific), rabbit anti-TNFR1 (clone H-271, 1:50, Santa Cruz), rabbit anti-TNFR2 (1:50, Sigma-Aldrich), and rat anti-IL-1Ra (clone 40,007, 1:1500, R&D Systems).

Techniques: Immunohistochemical staining, Staining

Journal: Life Science Alliance

Article Title: TRAP1 chaperone protein mutations and autoinflammation

doi: 10.26508/lsa.201900376

Figure Lengend Snippet: (A) PBMCs were isolated from TRAPS patients with different mutations and from three HCs (wild-type, WT), fixed, and labelled with the nuclear stain DAPI (blue), anti-TNFR1 antibodies (red), and anti-TRAP1 antibodies (green). Scale bar = 5 μm. (B) The degree of red-green co-localization was quantified in at least three images for each patient and from three HCs (WT). * P < 0.05 ** P < 0.01 (unpaired two-tailed t tests).

Article Snippet: These were fixed with 4% PFA for 20 min and then permeabilized with 0.5% triton X for 5 min. After this, nonspecific binding was blocked with donkey serum for 1 h. Coverslips were then incubated with mouse anti-TRAP1 (BD biosciences) and rabbit anti-TNFR1 (Santa-Cruz) for 45 min, and subsequently, Alexa Fluor-488 antimouse and Alexa Fluor-568 antirabbit secondary antibodies.

Techniques: Isolation, Staining, Two Tailed Test