biotinylated anti ym1  (R&D Systems)

Journal: bioRxiv

Article Title: Allergen-induced airway matrix remodelling in mice can be prevented or reversed by targeting chitinase-like proteins

doi: 10.1101/2023.08.18.553857

Figure Lengend Snippet: a) Representative images of lung tissue sections showing an airway from C57BL/6 or Il17a Cre Rosa26 eYFP female mice treated with PBS or DRA twice weekly for 8 weeks. Lung sections were stained with DAPI to visualise cell nuclei and antibodies recognising Ym1 and Ym2; scale bar=30μm. b) Analysis of staining intensity of Ym2 within airway epithelial cells normalised to airway basement membrane length or Ym2 and Ym2 staining intensity within regions of the lung parenchyma that exclude airways and large blood vessels from mice in a) . c) Representative images of lung tissue sections showing an airway from female Il13 +/+ or Il13 −/− mice treated with DRA twice weekly for 8 weeks. Lung sections were stained as in a) with scale bar=30μm. d) Ym1 and Ym2 staining intensity from from mice in c) analysed as in b) . mRNA expression of Chil1, Chil3 and Chil4 in whole lungs from e) C57BL/6 or Il17a Cre Rosa26 eYFP mice treated as in a) or f) Il13 eGFP wild-type, heterozygote or homozygote female mice treated with PBS or DRA twice weekly for 8 weeks. mRNA was relative to geometric mean of housekeeping genes Gapdh , Rpl13a and Rn45s . g) Representative flow cytometric identification of alveolar (Alv.Mo) and interstitial macrophages (Int.MØ) in Il13 eGFP mice treated as in f) . Gating based on Lineage − Ly6G − CD64 + MertK + cells within whole lung tissue digests with graphs showing the number of cells per gram of lung. h) The total numbers of numbers of Ym1 + alveolar (Alv.MØ) and interstitial macrophages (Int.MØ) per g of lung in Il13 eGFP mice treated as in f) and analysed by flow cytometry with representative flow cytometric plots show the percentage of Ym1 + or Ym1 − interstitial macrophages (Int.MØ). Datapoints show individual animals with bars representing mean ± s.e.m with n=2-9 female mice per group and images and flow plots are representative of n=7-8 mice per group. Data are from ( a,b and e ) one individual experiments or ( c, d, f-h ) two combined experiments and were analysed by ANOVA with Tukey’s multiple comparison test and significance level shown relative to PBS C57BL/6 or Il13 +/− mice or between C57BL/6 and Il17a Cre Rosa26 eYFP DRA mice or Il13 +/+ and Il13 −/− DRA mice as indicated on the graph. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 and NS, not significant.

Article Snippet: To measure intracellular RELM⍺ and Ym1, fixed cells were permeabilised with the Transcription Factor staining kit (eBioscience) for 20min at room temperature and stained for 45min with rabbit anti-mouse RELM α (Peprotech) and biotinylated goat anti-mouse Ym1 (R&D), followed by a 45min incubation with rabbit Alexa-Fluor 488 or AF594 Zenon labeling kit (Thermo) and Streptavidin PercP or BV421, BV785 or PeCy7.

Techniques: Staining, Membrane, Expressing, Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: Allergen-induced airway matrix remodelling in mice can be prevented or reversed by targeting chitinase-like proteins

doi: 10.1101/2023.08.18.553857

Figure Lengend Snippet: a) mRNA expression of Chil4, Chil1, Chil3 and Chia in whole lungs from littermate Chil4 KD mice treated with PBS or DRA twice weekly for 8 weeks. mRNA expression was relative to geometric mean of housekeeping genes Gapdh , Rpl13a and Rn45s. b) Secreted Ym2 levels in the BAL fluid of mice as in a) analysed by mass spectrometry; LFQ label free quantitation. c) Representative images of lung tissue sections from Chil4 WT/WT and Chil4 KD/KD mice treated with DRA as in a), stained with antibodies recognising Ym1 and Ym2 (scale bar=30μm). Graphs show analysis of staining intensity of Ym2 within airway epithelial cells normalised to airway basement membrane length or Ym1 and Ym2 staining intensity within regions of the lung parenchyma. d) Numbers of alveolar (Alv.MØ) and interstitial macrophages (Int.MØ) in whole lung tissue from mice as in a) analysed by flow cytometry. e) Representative histograms showing intracellular expression of Ym1 in Cd64 + MertK + SigF + Cd11c + Alv.MØ compared to isotype stained cells from mice as in a). Graph shows numbers of Ym1 + Alv.MØ per g of lung tissue. f) Representative flow cytometry plots of intracellular Ym1 expression in Int.MØ from mice treated as in a). Gates based on isotype staining for intracellular Ym1 and numbers show percentage of Ym1 + Int.MØ. Graph shows numbers of Ym1 + Int.MØ per g of lung tissue. Data for a-c are representative from 2 individual experiments (n=3-6 per group) and d-f from two combined experiments (n=4-12 female mice per group). Data were analysed by ANOVA with Tukey’s multiple comparison test and significance level shown relative to PBS Chil4 WT/WT mice or between Chil4 WT/WT and Chil4 KD/KD DRA mice as indicated on the graph. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 and NS, not significant.

Article Snippet: To measure intracellular RELM⍺ and Ym1, fixed cells were permeabilised with the Transcription Factor staining kit (eBioscience) for 20min at room temperature and stained for 45min with rabbit anti-mouse RELM α (Peprotech) and biotinylated goat anti-mouse Ym1 (R&D), followed by a 45min incubation with rabbit Alexa-Fluor 488 or AF594 Zenon labeling kit (Thermo) and Streptavidin PercP or BV421, BV785 or PeCy7.

Techniques: Expressing, Mass Spectrometry, Quantitation Assay, Staining, Membrane, Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: Allergen-induced airway matrix remodelling in mice can be prevented or reversed by targeting chitinase-like proteins

doi: 10.1101/2023.08.18.553857

Figure Lengend Snippet: a) BALB/c mice were treated with PBS and DRA intranasally twice weekly for 8 weeks. At the 4 week time point mice were also treated intraperitoneally with either IgG2a or anti-Ym1 for 4 weeks. b) Numbers of neutrophils and eosinophils in the BAL of mice were analysed by flow cytometry. c ) Numbers of CD4 + TCRβ + , cells expressing intracellular cytokine IL-4, IL-13 or IL-17a and TCRƔδ + TCRβ − cells expressing intracellular IL-17A from single cell suspensions of lungs from mice in a) stimulated ex vivo with PMA/ionomycin prior to staining with antibodies and performing flow cytometry. d) Lung tissue sections were stained with PAS and numbers of PAS + airway epithelial cells counted per airway and normalised to length of the basement membrane from mice as in a ). e ) Lung tissue sections were stained with Masson’s trichrome and MT + area was analysed around the airway excluding vascular regions and normalised to basement membrane length of mice as in a ). f ) Representative images of lung tissue sections showing from mice as in a ) stained with DAPI to visualise cell nuclei and antibodies recognising Ym1 and Ym2 (scale bar=30μm). Graph shows analysis of staining intensity of Ym1 and Ym2 in the lung parenchyma and Ym2 within airway epithelial cells normalised to airway basement membrane length. g ) Representative images of lung tissue sections from BALB/c mice treated as in a) stained with DAPI to visualise cell nuclei and antibodies or binding proteins recognising ⍺SMA, vimentin or Collagen I and IV or collagen III and HA binding protein (HABP) (scale bar = 30μm). Graphs show analysis of positive-stained area around the airways for each antigen and normalised to length of basement membrane. Data are from b, c and e are pooled from 2 individual experiments (n=8 per group) and data from d, f and g are representative of 2 individual experiments (n=5 per group). Datapoints show individual animals with bars representing mean ± s.e.m. Data were analysed by ANOVA with Tukey’s multiple comparison test and significance level shown relative to PBS IgG BALB/c mice or between IgG treated DRA mice and anti-Ym1 DRA treated mice as indicated on the graph. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 and NS, not significant.

Article Snippet: To measure intracellular RELM⍺ and Ym1, fixed cells were permeabilised with the Transcription Factor staining kit (eBioscience) for 20min at room temperature and stained for 45min with rabbit anti-mouse RELM α (Peprotech) and biotinylated goat anti-mouse Ym1 (R&D), followed by a 45min incubation with rabbit Alexa-Fluor 488 or AF594 Zenon labeling kit (Thermo) and Streptavidin PercP or BV421, BV785 or PeCy7.

Techniques: Flow Cytometry, Expressing, Ex Vivo, Staining, Membrane, Binding Assay, Comparison