peroxidase goat polyclonal anti mouse  (Vector Laboratories)

Journal: Nature Immunology

Article Title: Cathepsin L-dependent positive selection shapes clonal composition and functional fitness of CD4 + T cells

doi: 10.1038/s41590-025-02182-y

Figure Lengend Snippet: a , Representative flow cytometry plots of thymocyte subsets and percentages ± s.e.m. of CD4SP cells in Ctsl +/+ and Ctsl ΔTEC mice reconstituted with BM from Rag1 −/− OT-II ( Ctsl +/+ , n = 9; Ctsl ΔTEC , n = 7) , Rag1 −/− AND ( Ctsl +/+ , n = 8; Ctsl ΔTEC , n = 6) , Rag1 −/− Dep ( Ctsl +/+ , n = 9; Ctsl ΔTEC , n = 7) or Rag1 −/− AD10 ( Ctsl +/+ , n = 9; Ctsl ΔTEC , n = 8) TCR transgenic donors. b , Representative flow cytometry plots of the thymus and percentages ± s.e.m. of CD8SP cells in Ctsl +/+ and Ctsl ΔTEC mice reconstituted with BM from OT-I Tg Rag1 −/− donors ( n = 5 each). c , Representative flow cytometry plots and MFI ± s.e.m. of CD5 expression in DP thymocytes from BM chimeras as in a , relative to cells selected in Ctsl +/+ chimeras; WT, wild-type. d , Representative flow cytometry plots and MFI ± s.e.m. of CD5 expression in CD4SP thymocytes from Rag1 −/− Ctsl +/+ AD10 ( n = 3), Rag1 −/− Ctsl +/+ Dep ( n = 5), Rag1 −/− Ctsl +/+ AND ( n = 4) and Rag1 −/− Ctsl +/+ OT-II ( n = 4) TCR transgenic mice relative to polyclonal CD4SP thymocytes ( n = 3). P values in a and b were determined by Student’s two-tailed t -test and in c by Welch’s two-tailed t -test.

Article Snippet: Following protein transfer, the membrane was incubated with goat anti-mouse CTSL polyclonal IgG (AF1515, R&D; 1 μg ml –1 ), followed by secondary mouse anti-goat horseradish peroxidase-conjugated polyclonal IgG (205-035-108, Jackson ImmunoResearch; 40 ng ml –1 ) and SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).

Techniques: Flow Cytometry, Transgenic Assay, Expressing, Two Tailed Test

Journal: Nature Immunology

Article Title: Cathepsin L-dependent positive selection shapes clonal composition and functional fitness of CD4 + T cells

doi: 10.1038/s41590-025-02182-y

Figure Lengend Snippet: a , Pie charts representing LLO-specific clonotypes in the fully polyclonal repertoire of Tcra +/− Ctsl +/+ mice ( n = 316 TCRs) and Tcra +/− Ctsl ΔTEC mice ( n = 156 TCRs), identified by single-cell Tcra and Tcrb sequencing of sorted LLO-Tet + cells ( n ≥ 42 mice per genotype; clonotypes aggregated across 19 experiments). Segments in shades of gray represent TCRs exclusively found in one genotype; colored segments represent ‘public’ TCRs shared between genotypes. Segment size is proportional to the number of mice in which a given TCR was detected. b , Representative flow cytometry plots and percent ± s.e.m. of thymocyte subsets in Ctsl +/+ Lm54 Tg ( n = 3) and Ctsl ΔTEC Lm54 Tg mice ( n = 4; hereafter Ctsl ΔTEC Lm54 Tg and Ctsl +/+ Lm54 Tg , respectively). c , Representative flow cytometry plots and percent ± s.e.m. of thymocyte subsets in Rag1 −/− Ctsl +/+ Lm6 Tg ( n = 7) and Rag1 −/− Ctsl ΔTEC Lm6 Tg mice ( n = 9; hereafter Ctsl ΔTEC Lm6 Tg and Ctsl +/+ Lm6 Tg , respectively). d , Representative flow cytometry plots and number ± s.e.m. of lymph node CD4 + T cells in Ctsl +/+ Lm54 Tg ( n = 4) and Ctsl ΔTEC Lm54 Tg mice ( n = 5). e , Representative flow cytometry plots and number ± s.e.m. of lymph node CD4 + T cells in Ctsl +/+ Lm6 Tg ( n = 7) and Ctsl ΔTEC Lm6 Tg mice ( n = 9). f , Representative flow cytometry plots of MHCI and CD69 surface expression on CD4SP cells from Ctsl +/+ Lm54 Tg ( n = 9) and Ctsl ΔTEC Lm54 Tg mice ( n = 10). The percent ± s.e.m. of SM, M1 and M2 cells is indicated. g , Nur77 and surface CD5 expression (MFI ± s.e.m.) at consecutive DP and CD4SP stages of differentiation in Ctsl +/+ Lm54 Tg ( n = 4 or 5) and Ctsl ΔTEC Lm54 Tg ( n = 4 or 5) mice, assessed by intracellular staining and flow cytometry (Nur77) or flow cytometry (CD5). h , CD5 surface expression (MFI ± s.e.m.) at consecutive DP and CD4SP cell differentiation stages in Ctsl +/+ Lm6 Tg ( n = 4) and Ctsl ΔTEC Lm6 Tg mice ( n = 5), as assessed by flow cytometry. P values in b and c were determined by two-way ANOVA and a Sidak’s test for multiple comparisons. Data in d and e were analyzed by Student’s two-tailed t -test.

Article Snippet: Following protein transfer, the membrane was incubated with goat anti-mouse CTSL polyclonal IgG (AF1515, R&D; 1 μg ml –1 ), followed by secondary mouse anti-goat horseradish peroxidase-conjugated polyclonal IgG (205-035-108, Jackson ImmunoResearch; 40 ng ml –1 ) and SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).

Techniques: Sequencing, Flow Cytometry, Expressing, Staining, Cell Differentiation, Two Tailed Test

Journal: Nature Immunology

Article Title: Cathepsin L-dependent positive selection shapes clonal composition and functional fitness of CD4 + T cells

doi: 10.1038/s41590-025-02182-y

Figure Lengend Snippet: a , Cell size (Mean ± SEM) of M2 thymocytes in Ctsl +/+ Rag 1 −/− Lm54 Tg ( n = 3) and Ctsl ΔTEC Rag 1 −/− Lm54 Tg mice ( n = 4). b , CD44 surface expression (MFI ± SEM) at consecutive DP and CD4SP differentiation stages in Ctsl +/+ Rag 1 −/− Lm54 Tg ( n = 4) and Ctsl ΔTEC Rag 1 −/− Lm54 Tg mice ( n = 5); assessed by flow cytometry. c , CD69 and CD25 expression (MFI ± SEM) in polyclonal M2 thymocytes from Ctsl ΔTEC (n = 3) or Ctsl +/+ mice (n = 3) after 40 h culture with or without (unstim.) plate-bound antibodies to CD3 and CD28; assessed by flow cytometry. d , Representative flow cytometry plots of intracellular phospho-ERK staining of M2 thymocytes from Ctsl +/+ Rag 1 −/− Lm54 Tg ( n = 3) and Ctsl ΔTEC Rag 1 −/− Lm54 Tg mice (n = 3) after 5 min stimulation with PMA. Controls without stimulation define background. e , Donor cell ratio (Mean) in the spleen of 4.5 Gy-irradiated recipients ( n = 2) after transfer of a 1:1 mixture of CTV-labelled Ctsl +/+ and Ctsl ΔTEC M2 CD4SPs; assessed by flow cytometry. f , Replication index of donor cells on day 21 after transfer as in (e) assessed with FlowJo_v10.9.0 software, assessed by flow cytometry on the basis of CTV-dilution profiles. P-values by Student’s two-tailed t-test.

Article Snippet: Following protein transfer, the membrane was incubated with goat anti-mouse CTSL polyclonal IgG (AF1515, R&D; 1 μg ml –1 ), followed by secondary mouse anti-goat horseradish peroxidase-conjugated polyclonal IgG (205-035-108, Jackson ImmunoResearch; 40 ng ml –1 ) and SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).

Techniques: Expressing, Flow Cytometry, Staining, Irradiation, Software, Two Tailed Test