Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Lack of Major Histocompatibility Complex Class I Upregulation and Restrictive Infection by JC Virus Hamper Detection of Neurons by T Lymphocytes in the Central Nervous System

doi: 10.1097/nen.0000000000000218

Figure Lengend Snippet: FIGURE 1. T cells are present in white matter (WM), gray-white junction (GWJ), gray matter (GM), cerebellar granular layer (GL), and molecular layer (ML) areas harboring JCV-infected cells. Triple (A) and quadruple (BYD) immunostaining assays for CD8 (brown), CD3 (gray blue), T antigen (T Ag, purple), and VP1 (cyan-blue) show that numerous CD8-positive T cells (brown, filled circle arrow) are present in the vicinity of JC virus (JCV)Yinfected cells expressing T Ag (purple, arrowheads) in partially burnt out lesions located in the GWJ (A) square 1, magnified in inset 1 of an HIV-seropositive patient with progressive multifocal leukoencephalopathy. Rare presumed CD4-positive T cells (CD3 positive/CD8 negative, gray blue, asterisk in inset 1) are also present. The PML lesions extend partially in the GM (A, square 2, magnified in inset 2) where cells expressing T Ag can be seen (purple, arrowheads) as well as a few CD8-positive T cells located in the vessels (brown, empty circle arrows, inset 2). In B, the presence of CD8-positive and frequent presumed CD4-positive T cells in the GM of a JCV encephalopathy (JCVE) patient harboring numerous JCV-infected cells expressing T Ag and VP1. Numerous CD8-positive T cells in the parenchyma and blood vessels (brown, filled and empty circle arrows, respectively), presumed CD4-positive T cells (gray blue, asterisk), and JCV-infected cells expressing T Ag (purple, arrowhead) or VP1 (cyan-blue, arrow) are visible in the inset. An isolated cerebellar lesion at the border of the WM and GL of a JCV granule cell neuronopathy (JCV GCN) patient with chronic lymphocytic leukemia (C) and the magni- fication of the square in D show that CD8-positive T cells (brown, filled circle arrows) are present in the parenchyma in areas with JCV-infected cells expressing T Ag (purple, arrowheads) and/or VP1 (cyan-blue, arrows). Aggregates of CD8 on JCV-infected cells expressing VP1 are also evident. Scale bars = (AYC) 500 Km; (D) 50 Km.

Article Snippet: PAB597 m NA JCV VP1 SV40 T Ag (v-300); sc-20800 r IgG JCV T Ag Santa Cruz Biotechnology, Santa Cruz, CA MAP-2; HM-2; M4403 m IgG1 Neurons Sigma, St. Louis, MO MAP-2; ab5622 r IgG Neurons Chemicon, Temecula, CA MAP-2; LS-B290 ch IgY Neurons Lifespan Bioscience, Seattle, WA MAP-2; ab5392 ch IgY Neurons Abcam, Cambridge, MA NeuN (FOX3); ab104225 r IgG Neurons Abcam NeuN (FOX3); ABN90P gp IgG Neurons EMD Millipore, Billerica, MA GFAP; M0761; 6 F2 m IgG1 Astrocytes Dako, Carpinteria, CA GFAP; Z0334 r IgG Astrocytes Dako GFAP; ab4674 ch IgY Astrocytes Abcam GFAP; ab90601 sh IgG Astrocytes Abcam GFAP; 173004 gp IgG Astrocytes Synaptic Systems, Göttingen, DE CNPase; C5922; 11-5B m IgG1 Myelin/Oligo Sigma CNPase; ab18527 r IgG Myelin/Oligo Abcam CNPase; ab50739 ch IgY Myelin/Oligo Abcam CNP; HPA023266 r IgG Myelin/Oligo Sigma CD8; 1A5; VP-C325 m IgG1 CD8 Vector Laboratories, Burlingame, CA CD8 (144B); ab17147 m IgG1 CD8 Abcam CD3; CD3-12; OASA11552 rat IgG CD3 Aviva Systems Biology, San Diego, CA CD3; CD3-12; MCA1477 rat IgG CD3 AbD Serotec, Raleigh, NC CD68; M0814; KP1 m IgG1 Monocytes/ Dako Macrophages Granzyme B (11 F1); VP-G812 m IgG2a Granzyme B Vector Laboratories Perforin (5B10); VP-P967 m IgG1 Perforin Vector Laboratories MHC I (F-3), sc-55582 m IgG2a MHC I Santa Cruz Biotechnology MHC I (H-300), sc-25619 r IgG MHC I Santa Cruz Biotechnology HLA-A (EP1395Y); ab52922 r IgG MHC I Abcam ch, chicken; CNP and CNPase, CNP, 2¶,3¶-cyclic-nucleotide 3¶-phosphodiesterase; GFAP, glial fibrillary acidic protein; JCV, JC virus; m, mouse; MAG, myelin-associated glycoprotein; MAP-2, microtubule-associated protein 2; MOG, myelin-oligodendrocyte glycoprotein; oligo, oligodendrocytes; r, rabbit; SV40 T Ag, Simian virus 40 T antigen.

Techniques: Infection, Immunostaining, Virus, Expressing, Isolation

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Lack of Major Histocompatibility Complex Class I Upregulation and Restrictive Infection by JC Virus Hamper Detection of Neurons by T Lymphocytes in the Central Nervous System

doi: 10.1097/nen.0000000000000218

Figure Lengend Snippet: FIGURE 3. CD8-positive T cells aggregate mostly around cells with intact nuclei, expressing JC virus (JCV) T antigen (T Ag) and VP1. (A, B) Quadruple immunostaining for CD8 (brown), CD3 (gray blue), T Ag (purple), and VP1 (cyan-blue) done on an HIV- seronegative PML patient with chronic lymphocytic leukemia (A) and an HIV-seropositive PML patient (B). In PML lesions of the cerebral gray-white junction (GWJ) and gray matter (GM) (A, inset), where JCV-infected cells expressing VP1 are present (cyan-blue arrows), there are numerous parenchymal CD8-positive T cells (brown, black circle arrow). They are located mostly at the leading edge of infection where newly infected cells have still intact nuclei and express T Ag alone (purple, arrowhead) or T Ag and VP1 (cyan-blue arrows). In JCV-infected cerebellum (B), only rare CD8-positive T cells (brown, filled circle arrow) colocalize in the GL with hundreds of JCV-infected cells expressing T Ag (inset, purple, arrowheads), whereas more numerous CD8-positive T cells and some CD4-positive T cells (gray-blue, asterisk) colocalize in the contiguous WM with a few JCV-infected cells expressing VP1 (cyan-blue, arrow). (CYE) Triple immunofluorescence (IFA) staining for CD3, T Ag, and VP1 done in the cerebrum of an HIV-seronegative PML patient (C), an HIV-seropositive PML patient (D), and a JCV encephalopathy (JCVE) patient (E). The IFA immunostains confirm that T cells are absent in an area with T Ag only (C), whereas T cells are mostly present in areas where JCV-infected cells have intact nuclei with coexpression of T Ag and VP1 (pink, arrows in squares in D and E). Separate color channels are shown in insets. Scale bars = (A, B) 250 Km; (CYE) 100 Km.

Article Snippet: PAB597 m NA JCV VP1 SV40 T Ag (v-300); sc-20800 r IgG JCV T Ag Santa Cruz Biotechnology, Santa Cruz, CA MAP-2; HM-2; M4403 m IgG1 Neurons Sigma, St. Louis, MO MAP-2; ab5622 r IgG Neurons Chemicon, Temecula, CA MAP-2; LS-B290 ch IgY Neurons Lifespan Bioscience, Seattle, WA MAP-2; ab5392 ch IgY Neurons Abcam, Cambridge, MA NeuN (FOX3); ab104225 r IgG Neurons Abcam NeuN (FOX3); ABN90P gp IgG Neurons EMD Millipore, Billerica, MA GFAP; M0761; 6 F2 m IgG1 Astrocytes Dako, Carpinteria, CA GFAP; Z0334 r IgG Astrocytes Dako GFAP; ab4674 ch IgY Astrocytes Abcam GFAP; ab90601 sh IgG Astrocytes Abcam GFAP; 173004 gp IgG Astrocytes Synaptic Systems, Göttingen, DE CNPase; C5922; 11-5B m IgG1 Myelin/Oligo Sigma CNPase; ab18527 r IgG Myelin/Oligo Abcam CNPase; ab50739 ch IgY Myelin/Oligo Abcam CNP; HPA023266 r IgG Myelin/Oligo Sigma CD8; 1A5; VP-C325 m IgG1 CD8 Vector Laboratories, Burlingame, CA CD8 (144B); ab17147 m IgG1 CD8 Abcam CD3; CD3-12; OASA11552 rat IgG CD3 Aviva Systems Biology, San Diego, CA CD3; CD3-12; MCA1477 rat IgG CD3 AbD Serotec, Raleigh, NC CD68; M0814; KP1 m IgG1 Monocytes/ Dako Macrophages Granzyme B (11 F1); VP-G812 m IgG2a Granzyme B Vector Laboratories Perforin (5B10); VP-P967 m IgG1 Perforin Vector Laboratories MHC I (F-3), sc-55582 m IgG2a MHC I Santa Cruz Biotechnology MHC I (H-300), sc-25619 r IgG MHC I Santa Cruz Biotechnology HLA-A (EP1395Y); ab52922 r IgG MHC I Abcam ch, chicken; CNP and CNPase, CNP, 2¶,3¶-cyclic-nucleotide 3¶-phosphodiesterase; GFAP, glial fibrillary acidic protein; JCV, JC virus; m, mouse; MAG, myelin-associated glycoprotein; MAP-2, microtubule-associated protein 2; MOG, myelin-oligodendrocyte glycoprotein; oligo, oligodendrocytes; r, rabbit; SV40 T Ag, Simian virus 40 T antigen.

Techniques: Expressing, Virus, Immunostaining, Infection, Staining

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Lack of Major Histocompatibility Complex Class I Upregulation and Restrictive Infection by JC Virus Hamper Detection of Neurons by T Lymphocytes in the Central Nervous System

doi: 10.1097/nen.0000000000000218

Figure Lengend Snippet: FIGURE 5. CD8-positive T cells colocalize with JC virus (JCV)Yinfected glia rather than neurons in mixed areas. (A) Triple immu- nostaining for CD8 (brown), microtubule-associated protein 2 ([MAP-2] gray blue), and T antigen (T Ag) (purple) of the cerebrum of a HIV-seropositive patient with progressive multifocal leukoencephalopathy (PML).CD8-positive T cells (brown, black circle arrow) are common in the gray-white junction (GWJ) area (inset 1) in the vicinity of JCV-infected glia (MAP-2 negative) expressing T Ag (purple, arrowhead). Inset 2 shows that the isolated JCV-infected cells, which express T Ag alone (purple, arrowheads) and are located in the area free of CD8-positive T cells of the gray matter (GM), are in fact neurons (gray blue, arrowheads, MAP-2 positive)., (B) Triple immunofluorescence (IFA) staining for VP1 (inset 1), MAP-2 (inset 2), and granzyme B (inset 3) in the cerebrum of an HIV-seropositive PML patient shows that the CD8-positive T cells of the GWJ produce granzyme B when they are in contact with productively JCV-infected glia (MAP-2 negative, arrows). (C) Triple immunostaining for granzyme B (brown), MAP-2 (gray blue), and T Ag (purple) of the cerebrum of a JCV encephalopathy (JCVE) patient showing a T cell (black circle arrow) releasing its granzyme B (double arrow) in a JCV-infected neuron. (D) Quadruple IFA for CD3, CD8, granzyme B, and VP1 done on the same patient shown in C show numerous CD3-positive/CD8-positive T cells (yellow-red, filled circle arrows) producing granzyme B (white plus sign) as well as some presumed CD4-positive T cells (CD3 positive/CD8 negative, asterisk) devoid of granzyme B near VP1- expressing cells. A granzyme-producing CD3-positive/CD8-positive T cell (full circle arrow) apposed to a JCV-infected cell of neuronal phenotype expressing VP1 (arrow) is visible at the right of the inset. WM, white matter. Scale bars = (A) 1 mm; (BYD) 50 Km.

Article Snippet: PAB597 m NA JCV VP1 SV40 T Ag (v-300); sc-20800 r IgG JCV T Ag Santa Cruz Biotechnology, Santa Cruz, CA MAP-2; HM-2; M4403 m IgG1 Neurons Sigma, St. Louis, MO MAP-2; ab5622 r IgG Neurons Chemicon, Temecula, CA MAP-2; LS-B290 ch IgY Neurons Lifespan Bioscience, Seattle, WA MAP-2; ab5392 ch IgY Neurons Abcam, Cambridge, MA NeuN (FOX3); ab104225 r IgG Neurons Abcam NeuN (FOX3); ABN90P gp IgG Neurons EMD Millipore, Billerica, MA GFAP; M0761; 6 F2 m IgG1 Astrocytes Dako, Carpinteria, CA GFAP; Z0334 r IgG Astrocytes Dako GFAP; ab4674 ch IgY Astrocytes Abcam GFAP; ab90601 sh IgG Astrocytes Abcam GFAP; 173004 gp IgG Astrocytes Synaptic Systems, Göttingen, DE CNPase; C5922; 11-5B m IgG1 Myelin/Oligo Sigma CNPase; ab18527 r IgG Myelin/Oligo Abcam CNPase; ab50739 ch IgY Myelin/Oligo Abcam CNP; HPA023266 r IgG Myelin/Oligo Sigma CD8; 1A5; VP-C325 m IgG1 CD8 Vector Laboratories, Burlingame, CA CD8 (144B); ab17147 m IgG1 CD8 Abcam CD3; CD3-12; OASA11552 rat IgG CD3 Aviva Systems Biology, San Diego, CA CD3; CD3-12; MCA1477 rat IgG CD3 AbD Serotec, Raleigh, NC CD68; M0814; KP1 m IgG1 Monocytes/ Dako Macrophages Granzyme B (11 F1); VP-G812 m IgG2a Granzyme B Vector Laboratories Perforin (5B10); VP-P967 m IgG1 Perforin Vector Laboratories MHC I (F-3), sc-55582 m IgG2a MHC I Santa Cruz Biotechnology MHC I (H-300), sc-25619 r IgG MHC I Santa Cruz Biotechnology HLA-A (EP1395Y); ab52922 r IgG MHC I Abcam ch, chicken; CNP and CNPase, CNP, 2¶,3¶-cyclic-nucleotide 3¶-phosphodiesterase; GFAP, glial fibrillary acidic protein; JCV, JC virus; m, mouse; MAG, myelin-associated glycoprotein; MAP-2, microtubule-associated protein 2; MOG, myelin-oligodendrocyte glycoprotein; oligo, oligodendrocytes; r, rabbit; SV40 T Ag, Simian virus 40 T antigen.

Techniques: Virus, Infection, Expressing, Isolation, Staining, Triple Immunostaining

Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Lack of Major Histocompatibility Complex Class I Upregulation and Restrictive Infection by JC Virus Hamper Detection of Neurons by T Lymphocytes in the Central Nervous System

doi: 10.1097/nen.0000000000000218

Figure Lengend Snippet: FIGURE 6. Major histocompatibility complex class I (MHC I) is upregulated in infiltrates and in parenchymal cells in JC virus (JCV)Yinfected areas. (AYF) Multiple immunofluorescence (IFA) staining images of CD3, MHC I, and VP1 (A, D, E), microtubule- associated protein 2 (MAP-2), MHC I, CD8, and VP1 (B, C), and CD68 and MHC I (F). A typical uninfected area in the temporal lobe of an HIV-seropositive patient with progressive multifocal leukoencephalopathy (PML) are shown in A and B. In those areas, cells expressing high level of MHC I are rare and restricted to vessels, parenchymal CD3-positive (A) and CD8-positive T cells (B) are absent and only rare CD8-positive T cells (B, empty circle arrow) are seen in blood vessels. Conversely, numerous cells expressing high levels of MHC I (red) are present in lesions of an HIV-seronegative patient with miliary PML (C) and a JCV encephalopathy (JCVE) patient (D) that harbor parenchymal CD8-positive (C) or CD3-positive (D) T cells (filled circle arrows) close to JCV-infected cells expressing VP1 (arrow and inset). Some of the cells expressing a high level of MHC I are infiltrates in the parenchyma and perivascular cuffs as shown by double positive for CD3-positive T cells and MHC I in a JCVE patient (white, E, inset) and for CD68-positive macrophages and MHC I in a pediatric HIV-seropositive PML patient (white, F, inset). Scale bar = 50 Km.

Article Snippet: PAB597 m NA JCV VP1 SV40 T Ag (v-300); sc-20800 r IgG JCV T Ag Santa Cruz Biotechnology, Santa Cruz, CA MAP-2; HM-2; M4403 m IgG1 Neurons Sigma, St. Louis, MO MAP-2; ab5622 r IgG Neurons Chemicon, Temecula, CA MAP-2; LS-B290 ch IgY Neurons Lifespan Bioscience, Seattle, WA MAP-2; ab5392 ch IgY Neurons Abcam, Cambridge, MA NeuN (FOX3); ab104225 r IgG Neurons Abcam NeuN (FOX3); ABN90P gp IgG Neurons EMD Millipore, Billerica, MA GFAP; M0761; 6 F2 m IgG1 Astrocytes Dako, Carpinteria, CA GFAP; Z0334 r IgG Astrocytes Dako GFAP; ab4674 ch IgY Astrocytes Abcam GFAP; ab90601 sh IgG Astrocytes Abcam GFAP; 173004 gp IgG Astrocytes Synaptic Systems, Göttingen, DE CNPase; C5922; 11-5B m IgG1 Myelin/Oligo Sigma CNPase; ab18527 r IgG Myelin/Oligo Abcam CNPase; ab50739 ch IgY Myelin/Oligo Abcam CNP; HPA023266 r IgG Myelin/Oligo Sigma CD8; 1A5; VP-C325 m IgG1 CD8 Vector Laboratories, Burlingame, CA CD8 (144B); ab17147 m IgG1 CD8 Abcam CD3; CD3-12; OASA11552 rat IgG CD3 Aviva Systems Biology, San Diego, CA CD3; CD3-12; MCA1477 rat IgG CD3 AbD Serotec, Raleigh, NC CD68; M0814; KP1 m IgG1 Monocytes/ Dako Macrophages Granzyme B (11 F1); VP-G812 m IgG2a Granzyme B Vector Laboratories Perforin (5B10); VP-P967 m IgG1 Perforin Vector Laboratories MHC I (F-3), sc-55582 m IgG2a MHC I Santa Cruz Biotechnology MHC I (H-300), sc-25619 r IgG MHC I Santa Cruz Biotechnology HLA-A (EP1395Y); ab52922 r IgG MHC I Abcam ch, chicken; CNP and CNPase, CNP, 2¶,3¶-cyclic-nucleotide 3¶-phosphodiesterase; GFAP, glial fibrillary acidic protein; JCV, JC virus; m, mouse; MAG, myelin-associated glycoprotein; MAP-2, microtubule-associated protein 2; MOG, myelin-oligodendrocyte glycoprotein; oligo, oligodendrocytes; r, rabbit; SV40 T Ag, Simian virus 40 T antigen.

Techniques: Immunopeptidomics, Virus, Staining, Expressing, Infection

Journal: British Journal of Pharmacology

Article Title: B 1 and B 2 kinin receptor blockade improves psoriasis‐like disease

doi: 10.1111/bph.15077

Figure Lengend Snippet: Analysis of the immune cell subset phenotype in the skin of mice lacking kinin receptors submitted to the IMQ‐induced psoriasis model. The number of (a) GR1+‐, (b) F4/80+‐, (c) CD3+‐, and (d) CD4+‐positive cells was quantified by flow cytometry in psoriatic skin lesion of WT and kinin receptor knockout mice treated daily with imiquimod for 6 days. Using FACSCanto™ flow cytometry, 500.000 events were acquired, and the results expressed in percentage. The values are presented as the mean ± SEM of six individual animals per group. (e) The percentage of GL3+‐positive cells in dorsal skin of WT and KO mice, data are means ± SEM of five individual animals per group. Symbols represent values for individual mouse and bars represent mean values for each group. No outliers were removed from the database. *P < .05, significantly different from the WT control group; # P < .05, significantly different from naive and WT imiquimod‐treated mice; one‐way ANOVA with the Newman–Keuls post hoc test

Article Snippet: Cat# sc‐53515, RRID:AB_783639), anti‐CD11b (isotype: IgG, rat monoclonal antibody, 1:300, BD Biosciences Cat# 557396, RRID:AB_396679), anti‐CD3 (isotype: IgG, rat monoclonal antibody,1:300, Santa Cruz Biotech.

Techniques: Flow Cytometry, Knock-Out

Journal: British Journal of Pharmacology

Article Title: B 1 and B 2 kinin receptor blockade improves psoriasis‐like disease

doi: 10.1111/bph.15077

Figure Lengend Snippet: Relevance of B1 and B2 kinin receptors to the immune response induced by imiquimod (IMQ). On Day 7 of the experimental protocol, dorsal skin samples from mice were collected and examined by immunofluorescence assay. Representative images immunostained for CD3+, CD4+, CD8+, and IL‐17 in the psoriatic lesions induced by imiquimod in WT mice, knockout mice (KOB1, KOB2, and KOB1B2), and WT mice treated with vehicle (DMSO 0.03%, v/v), SSR240612C (1.0 mg·kg−1), or FR173657 (30 mg·kg−1). Naive (untreated group) samples were collected and subjected to the same analysis. Kinin‐B1 and B2 receptors were evaluated in three non‐consecutive serial sections from five mice each. The white dotted lines indicate the basal membrane. The white arrows indicate some of the positively immunolabelled cells

Article Snippet: Cat# sc‐53515, RRID:AB_783639), anti‐CD11b (isotype: IgG, rat monoclonal antibody, 1:300, BD Biosciences Cat# 557396, RRID:AB_396679), anti‐CD3 (isotype: IgG, rat monoclonal antibody,1:300, Santa Cruz Biotech.

Techniques: Immunofluorescence, Knock-Out

Journal: British Journal of Pharmacology

Article Title: B 1 and B 2 kinin receptor blockade improves psoriasis‐like disease

doi: 10.1111/bph.15077

Figure Lengend Snippet: Analysis of kinin receptor participation in the immune response triggered by imiquimod (IMQ) in the skin. The number of (a) CD3+‐, (b) CD4+‐, and (c) CD8+‐positive cells was evaluated in the psoriatic lesion of imiquimod‐treated mice. (d) IL‐17 was also detected in the imiquimod‐induced, psoriatic skin of knockout and WT mice treated with the antagonists. Data are means ± SEM of eight (CD3+ T lymphocyte), seven (CD4+ T cell), eight (CD8+ T cells), and seven (IL‐17) individual animals each from two separate experiments. Positively immunolabelled cells were quantitated by counting fluorescent dots from three non‐consecutive serial sections per mouse. Symbols represent values for individual mouse and bars represent mean values for each group. No outliers were removed from the database. *P < .05, significantly different from the WT control group (for knockouts animals) or from the vehicle group (for animals treated with antagonists); # P < .05 between naive and WT imiquimod‐treated mice or between naive and vehicle‐treated mice; one‐way ANOVA with the Newman–Keuls post hoc test

Article Snippet: Cat# sc‐53515, RRID:AB_783639), anti‐CD11b (isotype: IgG, rat monoclonal antibody, 1:300, BD Biosciences Cat# 557396, RRID:AB_396679), anti‐CD3 (isotype: IgG, rat monoclonal antibody,1:300, Santa Cruz Biotech.

Techniques: Knock-Out

Journal: Pediatric research

Article Title: Effect of TNF-alpha on CD3-zeta and MHC-I in postnatal rat hippocampus.

doi: 10.1203/01.pdr.0000238246.74817.a0

Figure Lengend Snippet: Figure 1. Western immunoblotting of the CD3-. Hippocampal tissue from rats of three different age groups, EPN, weanling and adolescent, were used for Western blotting for CD3-. Quantification of the optical densitometry for four studies is demonstrated in the bar diagram. The protein expression of the EPN hippocampus was assigned a value of 100%, and the values are shown as percentage of EPN (% signal of EPN). (*p 0.001). Error bars refer to the SEM.

Article Snippet: The blots were blocked with 5% (wt/vol) nonfat dry milk in PBS containing 0.1% tween-20 for one hour at room temperature, incubated with primary polyclonal rabbit anti rat CD3- antibody (FL-163): sc-20919, Santa Cruz Biotechnology, at 1:1,000 dilution for one hour at room temperature; or with primary MAb OX-18, MCA 51 G (Serotec, UK) against rat MHC-I (25), which recognizes an epitope in the 3 domain of most rat MHC-I and MHC-I proteins (26), at [10 g/mL], for two hours at room temperature; washed with PBS/0.1% tween-20 (5 times 5 min) incubated with Donkey anti-rabbit IgG HRP secondary antibody (Amersham), diluted 1:2,000 or with Goat anti-mouse IgG HRP secondary antibody Star77 (Serotec, UK), at [1 g/mL] respectively for one hour at room temperature; washed with PBS/ 0.1% tween-20 (5 times 5 min).

Techniques: Western Blot, Expressing

Journal: Pediatric research

Article Title: Effect of TNF-alpha on CD3-zeta and MHC-I in postnatal rat hippocampus.

doi: 10.1203/01.pdr.0000238246.74817.a0

Figure Lengend Snippet: Figure 4. Effects of TNF- in vivo injections on CD3- protein expression. A representative Western blot and quantification of the optical densitometry for the effect of TNF- on the CD3- protein expression in the (A) EPN, (B) weanling, and (C) adolescent hippocampus. Bar graphs show quantification of the optical densitometry for 6 animals. (*p 0.001). The hippocampal protein expression of vehicle treated rats was assigned a value of 100%, and the values are shown as percentage of control (% signal of control). Error bars refer to the SEM.

Article Snippet: The blots were blocked with 5% (wt/vol) nonfat dry milk in PBS containing 0.1% tween-20 for one hour at room temperature, incubated with primary polyclonal rabbit anti rat CD3- antibody (FL-163): sc-20919, Santa Cruz Biotechnology, at 1:1,000 dilution for one hour at room temperature; or with primary MAb OX-18, MCA 51 G (Serotec, UK) against rat MHC-I (25), which recognizes an epitope in the 3 domain of most rat MHC-I and MHC-I proteins (26), at [10 g/mL], for two hours at room temperature; washed with PBS/0.1% tween-20 (5 times 5 min) incubated with Donkey anti-rabbit IgG HRP secondary antibody (Amersham), diluted 1:2,000 or with Goat anti-mouse IgG HRP secondary antibody Star77 (Serotec, UK), at [1 g/mL] respectively for one hour at room temperature; washed with PBS/ 0.1% tween-20 (5 times 5 min).

Techniques: In Vivo, Expressing, Western Blot, Control

Journal: PLoS ONE

Article Title: SOD3 Reduces Inflammatory Cell Migration by Regulating Adhesion Molecule and Cytokine Expression

doi: 10.1371/journal.pone.0005786

Figure Lengend Snippet: Open bars represent LacZ animals and black bars represent SOD3 animals. Infiltration of CD3+ lymphocytes was inhibited in SOD3 animals 10 days after injury remaining at the level seen at earlier 3-day time point (20× magnification).

Article Snippet: Ten micrometer sections were fixed in acetone and stained with rabbit anti-rat CD3 and mouse anti-rat CD68 (Serotec, Oxford, UK).

Techniques: