mouse hrp cd68 sc 20060 santa cruz  (Santa Cruz Biotechnology)

Journal: iScience

Article Title: IL-27 mediates immune response of pneumococcal vaccine SPY1 through Th17 and memory CD4 + T cells

doi: 10.1016/j.isci.2023.107464

Figure Lengend Snippet: IL-27Rα deficiency halts the protective effect of the SPY1 vaccine (A) Mice were intranasally immunized with SPY1 plus CT or PBS plus CT weekly for 1 month. On day 28, mice were intranasally challenged with S. pneumoniae strain 19F and specimens were collected for analysis on day 31. (B). Bacterial colonization in nasal lavage fluid of each group after 19F challenge (n = 5–7). One-way ANOVA. Mean ± SEM.∗p < 0.05, ∗∗p < 0.01. (C) One week following the last immunization, mice were challenged intranasally with S. pneumoniae strain D39 (5 × 10 7 CFU). The survival rates were observed for 21 days post challenge (n = 10). Log rank test.∗p < 0.05. (D) H&E and peroxidase immune staining for Ly6G (neutrophil marker) and CD68 (macrophage marker) in the indicated groups 3 days after 19F challenge. Representative slides are shown for each group (200 ×; scale bars = 100 μm). (E) Total inflammation score. One-way ANOVA. Mean ± SEM.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (F) Quantification of neutrophils surrounding bronchioles from (D). One-way ANOVA. Mean ± SEM.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (G) Quantification of macrophages surrounding bronchioles from (D). One-way ANOVA. Mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Tissue sections were also stained with polyclonal goat anti-mouse CD68 and Ly6G (servicebio) and detected using a streptavidin biotin immuno-peroxidase system.

Techniques: Staining, Marker

Journal: iScience

Article Title: IL-27 mediates immune response of pneumococcal vaccine SPY1 through Th17 and memory CD4 + T cells

doi: 10.1016/j.isci.2023.107464

Figure Lengend Snippet: The immune protective effect of SPY1 vaccine is independent of glycolysis (A) Titers of sIgA in saliva and IgG in serum (n = 7–8). T-test. Mean ± SEM. (B) H&E staining and peroxidase immune staining in lung tissues for Ly6G and CD68 in WT SPY1 vaccinated mice that were administered either PBS (control) or 2-deoxy-glucose (2-DG) 3 days after 19F challenge. Representative slides are shown for each group (200 ×, scale bars = 100 μm). Total inflammation score and quantification of neutrophils and macrophages surrounding bronchioles (n = 3–6). T-test. Mean ± SEM. (C) ELISA to quantify IL-17A protein levels in lung tissues (n = 6–7). T-test. Mean ± SEM. (D) Representative flow cytometric plots depicting proportions of Th17 cells in the indicated groups. (E) The proportions and numbers of Th17 cells in for the indicated groups determined by flow cytometry (n = 5). T-test. Mean ± SEM.

Article Snippet: Tissue sections were also stained with polyclonal goat anti-mouse CD68 and Ly6G (servicebio) and detected using a streptavidin biotin immuno-peroxidase system.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: iScience

Article Title: IL-27 mediates immune response of pneumococcal vaccine SPY1 through Th17 and memory CD4 + T cells

doi: 10.1016/j.isci.2023.107464

Figure Lengend Snippet:

Article Snippet: Tissue sections were also stained with polyclonal goat anti-mouse CD68 and Ly6G (servicebio) and detected using a streptavidin biotin immuno-peroxidase system.

Techniques: Purification, Recombinant, Lysis, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Acta Neuropathologica Communications

Article Title: Microglial CD68 and L-ferritin upregulation in response to phosphorylated-TDP-43 pathology in the amyotrophic lateral sclerosis brain

doi: 10.1186/s40478-023-01561-6

Figure Lengend Snippet: Immunohistochemistry panels for analysis of microglial changes in the human ALS and hTDP-43-ΔNLS ‘rNLS’ mouse brains

Article Snippet: Mouse IgG 3 anti-CD68 (1:200; Abcam, Ab783) , Goat anti-mouse IgG 3 AlexaFluor® 594 (1:500; ThermoFisher, A21155) , Rabbit anti-GFAP (1:2,000; DAKO, Z0334) , Goat anti-rabbit AlexaFluor® 594 (1:500; Jackson Laboratories, 111–586-144).

Techniques: Immunohistochemistry

Journal: Acta Neuropathologica Communications

Article Title: Microglial CD68 and L-ferritin upregulation in response to phosphorylated-TDP-43 pathology in the amyotrophic lateral sclerosis brain

doi: 10.1186/s40478-023-01561-6

Figure Lengend Snippet: Microglia increase expression of CD68 and Iba1 in the ALS motor cortex. Microglia were identified by combined labelling of all microglial markers from round 1 and the tissue-wide and single cell average intensities of each of these markers was measured and used to quantify changes in functional marker expression in ALS; L-ferritin ( A–C ), HLA-DR ( D–F ), CD68 ( G–I ), CD74 ( J–L ), and Iba1 ( M–O ). Immunolabelling intensity of each functional marker was measured within the microglial master mask and normalised to the area of the microglial master mask. The tissue-wide average intensities were compared between control and ALS cases in the motor cortex and hippocampus for each functional marker ( A, D, G, J, and M ). Data presented as mean ± SD; control n = 10 and ALS n = 9–10. To designate cells as either high- or low-expressing for each functional marker of interest (MOI high or MOI low ), total microglia from all control and all ALS cases were pooled and the distribution curve for each marker was generated ( B, E, H, K, and N ). The threshold for each marker was determined and each cell was designated as either MOI high or MOI low for each functional marker. The percentage of MOI high cells was compared between control and ALS cases in the motor cortex and hippocampus for each functional marker ( C, F, I, L, and O ). Data presented as mean ± SD; control n = 10 and ALS n = 9–10. MOI average intensities and MOI high percentages were compared between case groups with multiple Mann–Whitney tests and multiple comparisons were controlled for using a False Discovery Rate of 0.01, as determined by the two-stage step-up method of Benjamini, Krieger, and Yekutieli. Significance of differences between case groups: *p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001

Article Snippet: Mouse IgG 3 anti-CD68 (1:200; Abcam, Ab783) , Goat anti-mouse IgG 3 AlexaFluor® 594 (1:500; ThermoFisher, A21155) , Rabbit anti-GFAP (1:2,000; DAKO, Z0334) , Goat anti-rabbit AlexaFluor® 594 (1:500; Jackson Laboratories, 111–586-144).

Techniques: Expressing, Functional Assay, Marker, Generated, MANN-WHITNEY

Journal: Acta Neuropathologica Communications

Article Title: Microglial CD68 and L-ferritin upregulation in response to phosphorylated-TDP-43 pathology in the amyotrophic lateral sclerosis brain

doi: 10.1186/s40478-023-01561-6

Figure Lengend Snippet: Microglia express functional markers in the normal and ALS post-mortem human motor cortex and hippocampus. Two rounds of immunohistochemical labelling were used to visualise microglial, anatomical, and pathological markers in the motor cortex ( A–H ) and hippocampus ( I–P ) from human ALS (stage 4 shown) and neurologically normal control cases. Round 1 was comprised of microglial markers, including L-ferritin (green), HLA-DR (magenta), CD68 (red), CD74 (yellow), and Iba1 (cyan), with a Hoechst nuclear counterstain (blue) ( A-B, E–F, I–J, M–N ). Round 2 was comprised of pathological and anatomical markers, including pTDP-43 (green), NeuN (magenta), GFAP (red), and lectin (yellow) with a Hoechst nuclear counterstain (blue) ( C–D, G–H, K–L, O–P ). Images from rounds 1 and 2 were aligned using the Hoechst nuclear counterstain. Scale bars ( A, C, E, I, K, M, O ) = 100 µm and scale bars ( B, D, F, H, J, L, N, P ) = 20 µm

Article Snippet: Mouse IgG 3 anti-CD68 (1:200; Abcam, Ab783) , Goat anti-mouse IgG 3 AlexaFluor® 594 (1:500; ThermoFisher, A21155) , Rabbit anti-GFAP (1:2,000; DAKO, Z0334) , Goat anti-rabbit AlexaFluor® 594 (1:500; Jackson Laboratories, 111–586-144).

Techniques: Functional Assay, Immunohistochemical staining

Journal: Acta Neuropathologica Communications

Article Title: Microglial CD68 and L-ferritin upregulation in response to phosphorylated-TDP-43 pathology in the amyotrophic lateral sclerosis brain

doi: 10.1186/s40478-023-01561-6

Figure Lengend Snippet: Microglial cell density, pTDP-43, and GFAP load are increased in ALS. Microglial cell density was quantified from round 1 immunolabelling ( A and B ) and pTDP-43 load and astrogliosis was quantified from round 2 ( C–F ). Examples of immunofluorescent images and binary masks are taken from stage 4 ALS case, MN13 ( A, C , and E ). Total microglia were identified by creating separate microglial marker binary masks from HLA-DR, CD68, CD74, and Iba1, which were then combined to create a microglial marker master mask ( A ). Each object within this mask was considered a single microglial cell. The microglial cell density in the motor cortex and hippocampus was quantified and compared between control and ALS cases ( B ). Aggregates of pTDP-43 were detected, and binary masks of immunoreactivity were generated ( C ). The integrated intensity of pTDP-43 aggregates identified was quantified and normalised to total tissue are to generate a load measure. The pTDP-43 load was compared between control and ALS motor cortex and hippocampus ( D ). The area of GFAP immunoreactivity identified was quantified and normalised to total tissue are to generate a measure of astrogliosis ( E and F ). Data presented as mean ± SD; control n = 10 and ALS n = 9–10. Microglial densities and pathology loads were compared between case groups with multiple Mann–Whitney tests and multiple comparisons were controlled for using a False Discovery Rate of 0.01, as determined by the two-stage step-up method of Benjamini, Krieger, and Yekutieli. Significance of differences between case groups: ****p ≤ 0.0001, **p ≤ 0.01, *p ≤ 0.05

Article Snippet: Mouse IgG 3 anti-CD68 (1:200; Abcam, Ab783) , Goat anti-mouse IgG 3 AlexaFluor® 594 (1:500; ThermoFisher, A21155) , Rabbit anti-GFAP (1:2,000; DAKO, Z0334) , Goat anti-rabbit AlexaFluor® 594 (1:500; Jackson Laboratories, 111–586-144).

Techniques: Marker, Generated, MANN-WHITNEY

Journal: Acta Neuropathologica Communications

Article Title: Microglial CD68 and L-ferritin upregulation in response to phosphorylated-TDP-43 pathology in the amyotrophic lateral sclerosis brain

doi: 10.1186/s40478-023-01561-6

Figure Lengend Snippet: Percentage of CD68 high microglia correlates with pTDP-43 load in the ALS motor cortex. Measures of microglial functional changes and neuropathology load were correlated in the control ( A-B ) and ALS ( C-D ) motor cortex and hippocampus. Measures of pTDP-43 integrated intensity and GFAP area coverage, and percentages of L-ferritin high , HLA-DR high , CD68 high , CD74 high , and Iba1 high microglia were sequentially correlated with one another using Spearman correlations. The resulting r value from each correlation is presented in the correlation matrix and colour coded relative to strength. All r values greater than 0.7 were statistically significant and considered very strong correlations. Control n = 10 and ALS n = 9–10, where statistical significance was set at p ≤ 0.05

Article Snippet: Mouse IgG 3 anti-CD68 (1:200; Abcam, Ab783) , Goat anti-mouse IgG 3 AlexaFluor® 594 (1:500; ThermoFisher, A21155) , Rabbit anti-GFAP (1:2,000; DAKO, Z0334) , Goat anti-rabbit AlexaFluor® 594 (1:500; Jackson Laboratories, 111–586-144).

Techniques: Functional Assay

Journal: Acta Neuropathologica Communications

Article Title: Microglial CD68 and L-ferritin upregulation in response to phosphorylated-TDP-43 pathology in the amyotrophic lateral sclerosis brain

doi: 10.1186/s40478-023-01561-6

Figure Lengend Snippet: Microglial clusters enriched ALS motor cortex express high levels of L-ferritin. Microglial single-cell average intensities of L-ferritin, HLA-DR, CD68, CD74, and Iba1 were used to identify and characterise microglial subpopulations that were changed in ALS relative to controls. A t-SNE plot was generated using random subset of 124,800 microglia (31,200 each from control motor cortex, ALS motor cortex, control hippocampus, and ALS hippocampus), and 27 clusters were identified using a k nearest neighbour and Louvain clustering approach ( A ). The total contribution of control versus ALS microglia to each cluster was visualised with a percentage bar to determine clusters most different between control and ALS: clusters 2, 4, 6, 11, and 19 ( B 1 -F 1 ). The percentage of total microglia in clusters 2, 4, 6, 11, and 19 were statistically compared between control and ALS motor cortex and hippocampus ( B 2 -F 2 ). Data presented as mean ± SD; control n = 10 and ALS n = 9–10. Cluster percentages were compared between case groups with multiple Mann–Whitney tests and multiple comparisons were controlled for using a False Discovery Rate of 0.01, as determined by the two-stage step-up method of Benjamini, Krieger, and Yekutieli. Significance of differences between case groups: *p ≤ 0.05. Microglial single-cell average intensities of L-ferritin, HLA-DR, CD68, CD74, and Iba1 were mapped on the merged t-SNE plot (all microglia from ALS and control motor cortex and hippocampus) using a binary high-low approach to visualise MOI low and MOI high microglia ( G ). To phenotype clusters based on MOI high percentage, for each functional marker the percentage of MOI high microglia in each cluster for a given case to the percentage of MOI high microglia in that case overallThe percentage MOI high for each functional marker was normalised to the total percentage of MOI high in each case for clusters 2, 4, 6, 11, and 19 in all cases ( H–L ). Data presented as mean ± SD; n = 20. Presence of CD68 high L-ferritin high ‘cluster 2’ microglia confirmed in stage 4 ALS case, MN15 ( M ). Representative image is a confocal Z -projection of Hoechst (blue; M 1 ), L-ferritin (green; M 2 ), CD68 (red; M 3 ), and Iba1 (cyan; M 4 ) co-labelling; scale bar = 20 µm

Article Snippet: Mouse IgG 3 anti-CD68 (1:200; Abcam, Ab783) , Goat anti-mouse IgG 3 AlexaFluor® 594 (1:500; ThermoFisher, A21155) , Rabbit anti-GFAP (1:2,000; DAKO, Z0334) , Goat anti-rabbit AlexaFluor® 594 (1:500; Jackson Laboratories, 111–586-144).

Techniques: Generated, MANN-WHITNEY, Functional Assay, Marker

Journal: Acta Neuropathologica Communications

Article Title: Microglial CD68 and L-ferritin upregulation in response to phosphorylated-TDP-43 pathology in the amyotrophic lateral sclerosis brain

doi: 10.1186/s40478-023-01561-6

Figure Lengend Snippet: Microglial CD68 and L-ferritin expression follow pTDP-43 aggregation in motor cortex and hippocampus of rNLS mice. Immunohistochemical labelling was used to visualise microglial, anatomical, and pathological markers in the of bigenic NEFH -tTA/ tetO -hTDP-43∆NLS (rNLS) and single transgenic tetO -hTDP-43∆NLS (control) mice at 2, 4, and 6 weeks off DOX. Example images of the motor cortex ( A–D ) and hippocampus ( E–J ) from mice at 6 weeks of DOX are shown; microglia were labelled with CD68 (green), L-ferritin (magenta), and Iba1 (red), pathology with pTDP-43 (yellow), and neurons with NeuN (white), with a Hoechst nuclear counterstain (blue); scale bar A = 100 µm and scale bar B = 20 µm. Microglial cell density ( K ), pTDP-43 load ( L ), and the percentage of CD68 high ( M ) and L-ferritin high ( N ) microglia were quantified in the hippocampus and motor cortex of control and rNLS mice 2, 4, and 6 weeks off DOX ( K ). All data presented as mean ± SD; control n = 4–6 and rNLS n = 2–5. Measurements were compared between case groups at each time point with a 2-way ANOVA and multiple comparisons were corrected for using Bonferroni’s multiple comparisons test. Significance of differences between case groups: *p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001

Article Snippet: Mouse IgG 3 anti-CD68 (1:200; Abcam, Ab783) , Goat anti-mouse IgG 3 AlexaFluor® 594 (1:500; ThermoFisher, A21155) , Rabbit anti-GFAP (1:2,000; DAKO, Z0334) , Goat anti-rabbit AlexaFluor® 594 (1:500; Jackson Laboratories, 111–586-144).

Techniques: Expressing, Immunohistochemical staining, Transgenic Assay

Journal: Acta Neuropathologica Communications

Article Title: Microglial CD68 and L-ferritin upregulation in response to phosphorylated-TDP-43 pathology in the amyotrophic lateral sclerosis brain

doi: 10.1186/s40478-023-01561-6

Figure Lengend Snippet: Summary of microglial changes identified in the human ALS and mouse rNLS motor cortex and hippocampus. The human ALS motor cortex showed increased microglial expression of both Iba1 and CD68, the latter of which strongly correlated with increased pTDP-43 load. Microglial phenotype clusters that were enriched in the ALS motor cortex were defined by high L-ferritin expression. In the rNLS mouse-driven model of ALS, higher microglial CD68 and L-ferritin expression occurred at early and late disease stages, respectively, after pTDP-43 aggregation

Article Snippet: Mouse IgG 3 anti-CD68 (1:200; Abcam, Ab783) , Goat anti-mouse IgG 3 AlexaFluor® 594 (1:500; ThermoFisher, A21155) , Rabbit anti-GFAP (1:2,000; DAKO, Z0334) , Goat anti-rabbit AlexaFluor® 594 (1:500; Jackson Laboratories, 111–586-144).

Techniques: Expressing