Journal: Nature Protocols

Article Title: Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope

doi: 10.1038/nprot.2014.155

Figure Lengend Snippet: Figure 3 | The components of the mmTIRF system and their assembly. (a) A diagram of the mmTIRF system (Mad City Labs) showing how each of the components interface. The system is designed around an objective holder fixed to a metal plate. A Nano-View/M 200-3 embedded-style xy-micrometer stage is then attached to this same plate. The nanopositioner itself is recessed into the micropositioner. A z-positioner with a dampening mass and a clamp to hold the U-shaped microscope slide holder is secured to the top plate of the nanopositioner. (b) The assembled micromirror TIRF system showing the objective platform, stages, legs and micromirror platform assemblies.

Article Snippet: Z488RDC, Z532RDC and ZT594RDC) Doublet lens for final focusing/steering lens (L3; Newport Corporation, cat. no. PAC067AR.14) Optical support rod for mounting He-Ne laser (Newport Corporation, cat. no. 70) Rack-and-pinion rod clamp part of EM-CCD camera mount (Newport Corporation, cat. no. 370-RC) Cylindrical laser mount (Newport Corporation, cat. no. ULM-TILT) Stainless steel optical posts for mounting optical components and lasers, 1/2-inch diameter (Thorlabs, TR series posts) Post holders for mounting optical components and lasers, 1/2-inch diameter (Thorlabs, PH series post holders) Post-holder bases (Thorlabs BA series) Kinematic cage mounts for mounting xy-translation mounts containing doublet lenses or objectives (Thorlabs, cat. no. KC1-T (×9)) xy-translation mounts for housing doublet lenses and objectives (Thorlabs, cat. no. LM1XY (9×)) Linear stage for fine translation mounted doublet lenses (Newport, cat. no. 423 (5×)) Rotation stage for mounting wave plates (Newport, cat. no. RSP-1T (6); Melles Griot, cat. no. 1100-10 (2×)) Kinematic platform mounts for mounting broadband polarizing cubes (Thorlabs, cat. no. KM100B (4×)) Clamping arm for mounting broadband polarizing cubes (Thorlabs, cat. no. PM3 (4×)) Precision kinematic mirror mounts (Newport Corporation, 1-inch diameter, cat. no. U100-A3K (6×)) Kinematic prism mount for mounting dichroic mirror clamps (Thorlabs, cat. no. KM100PM (3×)) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Plate holder for mounting dichroic mirror (Thorlabs, cat. no. FP01 (3×)) Post-mounted iris diaphragms (I2; Thorlabs, cat. no. ID25 (3×)) Polarizing sheet plate (Thorlabs, cat. no. LPVIS100) Shearing interferometer (Thorlabs, cat. no. SI100) Objective and micromirror platforms Olympus 60× 1.45-NA PlanApo Objective (MO) mmTIRF system (Mad City Labs) containing the following components: shelf braces (cat. no. 629065B (4×)); SM1-threaded ports/side plate (cat. no. 629075B (2×)); RM21 legs (cat. no. 628415 (4×)); sample holder (made from part nos.

Techniques: Microscopy

Journal: Nature Protocols

Article Title: Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope

doi: 10.1038/nprot.2014.155

Figure Lengend Snippet: Figure 5 | A diagram of the position of the components of the microscope on the optical table.

Article Snippet: Z488RDC, Z532RDC and ZT594RDC) Doublet lens for final focusing/steering lens (L3; Newport Corporation, cat. no. PAC067AR.14) Optical support rod for mounting He-Ne laser (Newport Corporation, cat. no. 70) Rack-and-pinion rod clamp part of EM-CCD camera mount (Newport Corporation, cat. no. 370-RC) Cylindrical laser mount (Newport Corporation, cat. no. ULM-TILT) Stainless steel optical posts for mounting optical components and lasers, 1/2-inch diameter (Thorlabs, TR series posts) Post holders for mounting optical components and lasers, 1/2-inch diameter (Thorlabs, PH series post holders) Post-holder bases (Thorlabs BA series) Kinematic cage mounts for mounting xy-translation mounts containing doublet lenses or objectives (Thorlabs, cat. no. KC1-T (×9)) xy-translation mounts for housing doublet lenses and objectives (Thorlabs, cat. no. LM1XY (9×)) Linear stage for fine translation mounted doublet lenses (Newport, cat. no. 423 (5×)) Rotation stage for mounting wave plates (Newport, cat. no. RSP-1T (6); Melles Griot, cat. no. 1100-10 (2×)) Kinematic platform mounts for mounting broadband polarizing cubes (Thorlabs, cat. no. KM100B (4×)) Clamping arm for mounting broadband polarizing cubes (Thorlabs, cat. no. PM3 (4×)) Precision kinematic mirror mounts (Newport Corporation, 1-inch diameter, cat. no. U100-A3K (6×)) Kinematic prism mount for mounting dichroic mirror clamps (Thorlabs, cat. no. KM100PM (3×)) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Plate holder for mounting dichroic mirror (Thorlabs, cat. no. FP01 (3×)) Post-mounted iris diaphragms (I2; Thorlabs, cat. no. ID25 (3×)) Polarizing sheet plate (Thorlabs, cat. no. LPVIS100) Shearing interferometer (Thorlabs, cat. no. SI100) Objective and micromirror platforms Olympus 60× 1.45-NA PlanApo Objective (MO) mmTIRF system (Mad City Labs) containing the following components: shelf braces (cat. no. 629065B (4×)); SM1-threaded ports/side plate (cat. no. 629075B (2×)); RM21 legs (cat. no. 628415 (4×)); sample holder (made from part nos.

Techniques: Microscopy

Journal: Nature Protocols

Article Title: Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope

doi: 10.1038/nprot.2014.155

Figure Lengend Snippet: Figure 7 | The components and layout of the emission path optics. (a) Location of the micromirrors, iris and the 45° mirror under the microscope objective. (b) Mounting of the EM-CCD camera. (c) The dual-view optics as seen looking toward the EM-CCD camera. (d) The dual-view optics as viewed looking toward the microscope objective.

Article Snippet: Z488RDC, Z532RDC and ZT594RDC) Doublet lens for final focusing/steering lens (L3; Newport Corporation, cat. no. PAC067AR.14) Optical support rod for mounting He-Ne laser (Newport Corporation, cat. no. 70) Rack-and-pinion rod clamp part of EM-CCD camera mount (Newport Corporation, cat. no. 370-RC) Cylindrical laser mount (Newport Corporation, cat. no. ULM-TILT) Stainless steel optical posts for mounting optical components and lasers, 1/2-inch diameter (Thorlabs, TR series posts) Post holders for mounting optical components and lasers, 1/2-inch diameter (Thorlabs, PH series post holders) Post-holder bases (Thorlabs BA series) Kinematic cage mounts for mounting xy-translation mounts containing doublet lenses or objectives (Thorlabs, cat. no. KC1-T (×9)) xy-translation mounts for housing doublet lenses and objectives (Thorlabs, cat. no. LM1XY (9×)) Linear stage for fine translation mounted doublet lenses (Newport, cat. no. 423 (5×)) Rotation stage for mounting wave plates (Newport, cat. no. RSP-1T (6); Melles Griot, cat. no. 1100-10 (2×)) Kinematic platform mounts for mounting broadband polarizing cubes (Thorlabs, cat. no. KM100B (4×)) Clamping arm for mounting broadband polarizing cubes (Thorlabs, cat. no. PM3 (4×)) Precision kinematic mirror mounts (Newport Corporation, 1-inch diameter, cat. no. U100-A3K (6×)) Kinematic prism mount for mounting dichroic mirror clamps (Thorlabs, cat. no. KM100PM (3×)) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Plate holder for mounting dichroic mirror (Thorlabs, cat. no. FP01 (3×)) Post-mounted iris diaphragms (I2; Thorlabs, cat. no. ID25 (3×)) Polarizing sheet plate (Thorlabs, cat. no. LPVIS100) Shearing interferometer (Thorlabs, cat. no. SI100) Objective and micromirror platforms Olympus 60× 1.45-NA PlanApo Objective (MO) mmTIRF system (Mad City Labs) containing the following components: shelf braces (cat. no. 629065B (4×)); SM1-threaded ports/side plate (cat. no. 629075B (2×)); RM21 legs (cat. no. 628415 (4×)); sample holder (made from part nos.

Techniques: Microscopy

Journal: Journal of Advanced Research

Article Title: The effects of primary cilia-mediated mechanical stimulation on nestin + -BMSCs during bone-tendon healing

doi: 10.1016/j.jare.2024.09.012

Figure Lengend Snippet: (A to C) The transverse and longitudinal migration ability of nestin + -BMSCs were measured by scratch assay and transwell assay, respectively. (D) The proliferative activity of nestin + -BMSCs was detected by CCK8. (E & F) Alcian blue staining, alizarin red staining and gene expression levels detected by qRT-PCR were used to analyze the chondrogenic and osteogenic differentiation potential of nestin + -BMSCs. (G) The expression of primary cilia in nestin + -BMSCs was detected by ace-tubulin fluorescence staining. Bar indicated 20 μm. (H) Expression of actin in nestin + -BMSCs stained with TRITC labeled Phalloidin Rhodamine. Bar indicated 50 μm. (I) The expression of acive-YAP in nestin + -BMSCs was detected by immunofluorescence. Bar indicated 50 μm. (J) The expression of TAZ in nestin + -BMSCs was detected by immunofluorescence. Bar indicated 50 μm. (K & L) Expression of key components of Hippo signaling pathway in nestin + -BMSCs. *, p < 0.05; #, p < 0.01.

Article Snippet: The expression of IFT88 and key components of Hippo/YAP signaling pathway (p-LATS: CST, #8654; p-YAP: CST, #13008; active-YAP: CST, #29495; Total YAP: CST, #4912, p-TAZ: CST, #59971, TAZ: CST, #83669; 14–3-3 protein: CST, #8312) in nestin + -BMSCs of each group were detected by WB assay.

Techniques: Migration, Wound Healing Assay, Transwell Assay, Activity Assay, Staining, Gene Expression, Quantitative RT-PCR, Expressing, Fluorescence, Labeling, Immunofluorescence

Journal: Journal of Advanced Research

Article Title: The effects of primary cilia-mediated mechanical stimulation on nestin + -BMSCs during bone-tendon healing

doi: 10.1016/j.jare.2024.09.012

Figure Lengend Snippet: (A & B) Inhibitory effect of actin on active-YAP and TAZ expression in nestin + -BMSCs. Bar indicated 50 μm. (C & D) Inhibitory effect of actin on expression of key components of Hippo signaling pathway in nestin + -BMSCs. (E) The effect of inhibiting Hippo signaling pathway on nestin + -BMSCs’ proliferation activity was detected by CCK8. (F to H) The effects of Hippo signaling inhibition on nestin + -BMSCs’ migration were detected by scratch assay and transwell assay. (I & J) The effects of Hippo signaling inhibition on nestin + -BMSCs’ differentiation potential were detected by alcian blue staining, alizarin red staining and gene expression levels. *, p < 0.05; #, p < 0.01.

Article Snippet: The expression of IFT88 and key components of Hippo/YAP signaling pathway (p-LATS: CST, #8654; p-YAP: CST, #13008; active-YAP: CST, #29495; Total YAP: CST, #4912, p-TAZ: CST, #59971, TAZ: CST, #83669; 14–3-3 protein: CST, #8312) in nestin + -BMSCs of each group were detected by WB assay.

Techniques: Expressing, Activity Assay, Inhibition, Migration, Wound Healing Assay, Transwell Assay, Staining, Gene Expression

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement C3d enables cell-mediated immunity capable of distinguishing spontaneously transformed from nontransformed cells.

doi: 10.1073/pnas.2405824121

Figure Lengend Snippet: Fig. 1. C3d injection of Vk*myc mice decreases paraproteinemia. Vk*myc mice older and younger than 1 y with detected M-protein, were injected with 20 microg C3d peptide (N = 16) or PBS (N = 13) intratibia 2 mo before analysis. (A) and (B) Shown is the change in the blood IgG concentration in Vk*myc mice following C3d or control injection. (C) M protein (arrows) was detected by SPEP before and at the end of treatment. M protein, seen as a single narrow band in the gamma region of the gel decreases in mice treated with C3d. Instead, a broad band of polyclonal Ig appears. In control mice the narrow band increases relative to the albumin band. (D) The graph shows the area under the globulin curve comparing analysis of the blood proteins before and after treatment with C3d or with PBS. The units are arbitrary and were determined by selecting the respective areas in image J using the average between lower peaks as the Bottom line. (A) Comparisons were by the double-tailed paired t test. **, P < 0.01, ***, P < 0.001. (B) Comparison was by the two-tailed Mann–Whitney test. ****, P < 0.0001. (D) Comparisons were by the ratio paired T test, P = 0.03.

Article Snippet: Slides were incubated overnight at 4 °C with goat IgG anti- mouse C3d antibody, (R&D Systems Cat: AF2655), diluted 1:125 in PBST containing 0.1% BSA.

Techniques: Injection, Concentration Assay, Control, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement C3d enables cell-mediated immunity capable of distinguishing spontaneously transformed from nontransformed cells.

doi: 10.1073/pnas.2405824121

Figure Lengend Snippet: Fig. 2. C3d induces CMI that in turn depletes malignant cells. Vk*myc mice older and younger than 1 y, with detected M-protein, were injected with 20 μg C3d peptide (N = 6) or PBS (N = 6) intratibia 2 mo before analysis. M protein was detected by SPEP. To accelerate the development of Multiple Myeloma, Vk*myc mice were immunized three times with 4-hydroxy-3-nitrophenyl (NP) conjugated to ovalbumin (NP-OVAL) once every month starting 3 mo before treatment with C3d. (A) Tsne plots of mass cytometry (CYTOF) indicating the various cell clusters. (B) Typical CYTOF tsne plots of plasma cell clusters in purple and B cells (not plasma cells) in red. CYTOF analysis of a representative C57BL/6 mouse of comparable age to the plots of the Vk*myc mice is shown in the far right (Control without MM). (C) Frequency of plasma cells detected by CYTOF of bone marrow. (D–F) ELISPOT analysis of IgG antibody secreting cells (D) or of NP-specific IgG ASC (E) in BM, or of NP-specific B cells in the Spleen, SP (F), 2 mo after C3d peptide or with PBS injection intratibia. (G–J) Vk*myc mice treated as explained above were divided in two groups 7 d after C3d injection. One group was treated with Cyclosporin (CsA) (N = 5) administered at a concentration of 3 mg/kg twice weekly for 7 wk until end point, and another group treated with PBS (N = 12) 2 mo after C3d injection. (G and H) Serum IgG concentration before and after treatment. (I–J) Number of plasma cells determined by FACS counting CD19-, B220lo, CD138+ cells in 100,000 live cells in the bone marrow (I) or in the spleen (I). Analyses in (C–E) and (G–I) were by the double-tailed paired t test, reflecting paired samples. **, P < 0.01;***, P < 0.001. (F and J) Comparison was by the two-tailed Mann–Whitney test, reflecting nonpaired measurements. Significance was considered if P < 0.05.

Article Snippet: Slides were incubated overnight at 4 °C with goat IgG anti- mouse C3d antibody, (R&D Systems Cat: AF2655), diluted 1:125 in PBST containing 0.1% BSA.

Techniques: Injection, Mass Cytometry, Clinical Proteomics, Control, Enzyme-linked Immunospot, Concentration Assay, Comparison, Two Tailed Test, MANN-WHITNEY, IF-P

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement C3d enables cell-mediated immunity capable of distinguishing spontaneously transformed from nontransformed cells.

doi: 10.1073/pnas.2405824121

Figure Lengend Snippet: Fig. 3. C3d decreases malignant plasma cells sparing normal plasma cells. Vk*myc mice were injected with 20 microg C3d peptide (N = 6) or with PBS (N = 5) intratibia 2 mo before analysis. Malignancy and nonmalignancy associated gene expression was analyzed in 3,995 individual plasma cells obtained from the bone marrow. (A) UMAP plots of sc RNA gene expression depicting the different cell populations analyzed. (B) Graph depicts the differential expression (DE) of a set of malignancy associated or nonmalignancy associated genes in the upper and lower percentile 20 for each category and in each group of mice. Each horizontal line represents a gene, each vertical line represents a cell. (C) Graphs show PCA of the expression of selected genes in the upper and lower percentile 20 associated with malignancy or nonmalignancy in all the plasma cells analyzed from both C3d-treated and untreated mice. The genes chosen contributed most of the difference between malignant and nonmalignant cells. (D) Distribution of clones according to a compound score obtained by subtracting the malignant score value from the nonmalignant score value for each cell. Upper graph depicts a histogram with bins separated by 0.2 compound score units. Frequencies (Y-axis) are shown as a percent of the clones (obtained from C3d treated or controls) included in the analysis. Lower graph is a scatter plot of clones according to their compound score. Statistical analysis by the Mann–Whitney test yielded P < 0/0001, (****). (E) Two-dimensional plots depicting malignant (X- axis) and nonmalignant (Y-axis) gene expression scores from treated and untreated mice. The size and color of the circles represent the number of copies of each clonotype defined according to the CDR3 sequence, VH and JH identity.

Article Snippet: Slides were incubated overnight at 4 °C with goat IgG anti- mouse C3d antibody, (R&D Systems Cat: AF2655), diluted 1:125 in PBST containing 0.1% BSA.

Techniques: Clinical Proteomics, Injection, Gene Expression, Quantitative Proteomics, Expressing, Clone Assay, MANN-WHITNEY, Sequencing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement C3d enables cell-mediated immunity capable of distinguishing spontaneously transformed from nontransformed cells.

doi: 10.1073/pnas.2405824121

Figure Lengend Snippet: Fig. 4. C3d treatment decreases the frequency of highly mutated malignant B cell clones and Ig class switched B cells in the BM but increases the frequency of normal maturing B cells. Vk*myc mice were injected with 20 microg C3d peptide (N = 6) or with PBS intratibia 2 mo before analysis (N = 5). Single cell V(D) J sequences were obtained using the 10X genomics kits followed by NGS. IgH+L sequences were further analyzed using the ARGalaxy immune repertoire pipeline as well as the NCBI Ig Blast and IMGT suites. (A) Frequency distribution of Ig isotypes in Vk-MYC* mice treated or not (Unt) with C3d. Comparison of the frequencies of IgM-producing clones in the BM by the Mann–Whitney test of mice treated or not with C3d indicated P = 0.0157. IgG1 frequencies also differed according to the treatment (P = 0.0476 Mann–Whitney test). (B) IgVH family frequencies in B cells in the bone marrow of Vk-MYC* mice treated or not (Unt) with C3d. Differences in the frequencies according to treatment did not reach significance by multiple Mann–Whitney tests. (C and D) Typical Circos analysis (ARGalaxy immune repertoire pipeline) of the V-J recombination in H chains obtained from bone marrow of mice C3d treated (C) or not (D) with C3d. The ribbons represent the top most frequent VH/JH gene combinations and they are chosen by the program automatically. The width is proportional to the overall frequency of VH/ JH combinations. Around the circle the colored bars reflect each JH or VH. Figure Shows there are fewer colored ribbons in C3d-treated mice (C) as compared to untreated (D) mice that have wider and/or more frequent VH-JH ribbons, indicative of clonal skewing. (E) CDR3 AA length distribution curves obtained from bone marrow B cells of mice treated or not with C3d. Untreated CDR3 lengths are skewed and show a peak of 8 AA length. Comparison of the curves by the Wilcoxon matched ranks test indicates an exact P value of 0.0092. The area under the curves (AUC) is also indicated. (F) The graph shows the frequency of Ig clones (larger than 10 cells) with VH mutation frequencies above 2%. Each dot represents the average frequency of mutated clonotypes in all the V(D)J sequences obtained from one mouse marrow. Data analysis by the Mann–Whitney test indicated a P = 0.0317. (G). Number of mutations per VH region in heavy chain sequences obtained from B cells isolated from the bone marrow of Vk-MYC* mice treated or not (Unt) with C3d. Each dot represents mutation frequencies in VH sequences in one cell. Each clonotype is counted only once. Frequencies were analyzed by ANOVA followed by the Kruskal–Wallis multiple comparisons test. Comparisons that yielded P < 0.0001 are noted by ****.

Article Snippet: Slides were incubated overnight at 4 °C with goat IgG anti- mouse C3d antibody, (R&D Systems Cat: AF2655), diluted 1:125 in PBST containing 0.1% BSA.

Techniques: Clone Assay, Injection, Comparison, MANN-WHITNEY, Mutagenesis, Isolation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement C3d enables cell-mediated immunity capable of distinguishing spontaneously transformed from nontransformed cells.

doi: 10.1073/pnas.2405824121

Figure Lengend Snippet: Fig. 5. C3d induces clonal-specific deletion of antigen-specific MM cells. Vk*myc mice were immunized 3 times with 4-hydrox-3-nitrophenyl(acetyl) (NP) coupled to Ovalbumin with 2 mo intervals, starting at 8 to 10 wk. Mice were injected with 20 microg C3d peptide (N = 6) or with PBS (N = 5) intratibia 2 mo before analysis. Analysis of BM cells was by 10X genomics. (A) Graph depicts the frequency of VH1-72 usage in each mouse. VH1-72 is the main VH region recruited by NP antigens. Comparison was by the Mann–Whitney test, P = 0.0286. (B) The number of IgG or IgA VH1-72 sequences in clones obtained from mice treated or not with C3d was compared by Fisher’s Exact test, P < 0.0001. (C and D) Seq2Logo depicting the CDR1 aminoacid motifs in B cell clones with VH1-72 from mice treated (C) or not (D) with C3d. Stacks represent AA with the size of the letters proportional to their prevalence. Shown is the Kullback–Leibler Logo. AA below “0” depict depleted AA. Apparent is the depletion of L at position 8 (corresponding to AA33 in the VH sequence) in mice treated with C3d. The W33L mutation causes a 10X increase in affinity to the NP hapten. Seq2Logo is available at http://www.cbs.dtu.dk/biotools/Seq2Logo. (E) Graph depicts the size of each clone shown as the Log10 of the number of sequences in each clone (dot), (C3d N = 115; PBS N = 110). Clones were defined by 80% similarity in CDR3 AA, same VH and JH. Results were compared by the Mann–Whitney test, P = 0.0006. (F) Number of mutations per VH1-72 regions in heavy chain sequences obtained from B cells isolated from the bone marrow of Vk-MYC* mice treated or not with C3d. Each dot represents the VH mutation frequency/cell. Each clonotype is counted only once. Only VH regions with mutation frequencies above 10% are shown. Results were analyzed by ANOVA followed by the Kruskal–Wallis multiple comparisons test.

Article Snippet: Slides were incubated overnight at 4 °C with goat IgG anti- mouse C3d antibody, (R&D Systems Cat: AF2655), diluted 1:125 in PBST containing 0.1% BSA.

Techniques: Injection, Comparison, MANN-WHITNEY, Clone Assay, Sequencing, Mutagenesis, Isolation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement C3d enables cell-mediated immunity capable of distinguishing spontaneously transformed from nontransformed cells.

doi: 10.1073/pnas.2405824121

Figure Lengend Snippet: Fig. 6. C3d increases expression of genes encoding ribosomal proteins and Long Noncoding RNAs. Vk*myc mice were injected with 20 microg C3d peptide (N = 6) or with PBS (N = 5) intratibia 2 mo before single cell RNA analysis of bone marrow cells. (A and B) UMAP plots of sc RNA gene expression depicting the different cell populations analyzed indicating hMYC expression to identify cells of the B cell lineage that activated hMYC. (C) Genes differentially expressed in C3d-treated relative to untreated mice in all the bone marrow cell populations indicated. Scale on the right refers to the Log2 fold changes. (D) GSEA pathway analysis of differentially expressed genes in various bone marrow cell populations. Major pathways are identified below and the cell types on the Left. The normalized enrichment score is shown in a colored scale and the number of genes differentially expressed in each pathway are represented in proportion to the size of the circle.

Article Snippet: Slides were incubated overnight at 4 °C with goat IgG anti- mouse C3d antibody, (R&D Systems Cat: AF2655), diluted 1:125 in PBST containing 0.1% BSA.

Techniques: Expressing, Injection, Gene Expression

Journal: bioRxiv

Article Title: ERK5 is required for neutrophil-mediated ROS release and essential in epidermolysis bullosa acquisita

doi: 10.1101/2025.11.12.688045

Figure Lengend Snippet: (A) Lesional and healthy skin obtained from immunization-induced EBA and healthy control mice was immunohistochemically stained for phosphorylated ERK5 (pERK5). Scale bar represents 100 µM. The dermal-epidermal junction is indicated by the black lines. Arrows indicate stained cells. (B) Enhanced staining in both dermis and epidermis in lesional treated mice was observed in a semi-quantitative measurement. (C) Immortalized keratinocytes (HaCaT) were stimulated with anti-COL7 C IgG and C5a concentration was measured using ELISA, which showed no significant changes upon ERK5 inhibition. (D-F) Antibody transfer–induced EBA was induced by injection of rabbit anti-mCOL7 C antibodies in C57BL/6J mice, and prophylactic treatment with XMD8-92 twice daily significantly reduced clinical disease scores, affected ear surface area (AESA), and delta ear thickness (referring to day 0) compared to vehicle control. (G,H) In skin samples obtained on the final day of the antibody transfer–induced EBA model, histological analyses revealed no significant effects of ERK5 inhibition on leukocyte infiltration (asterisk) or dermal–epidermal split formation (arrows). IgG or C3 deposition remained unchanged (arrows). (I–J) Cryosections were stained for IgG and C3 deposition, which were both unaffected by ERK5 inhibition. B : n = 5, Tukey boxplot Mann-Whitney test, *p ≤ 0.05; C : n = 3,, mean ± SEM; E : n = 6 (control)/5 (XMD8-92), mean ± SEM, mixed effect analysis with Šidák’s multiple comparisons test, **p ≤ 0.01, ***p ≤0.001, ****p ≤ 0.0001; G-J : n = 6 (control)/5 (XMD8-92), Tukey boxplot, Mann-Whitney test

Article Snippet: C5a concentration in supernatants was determined using the Human Complement Component C5a DuoSet ELISA (R&D Systems GmbH, Wiesbaden, Germany) in accordance with manufacturer instructions and measured in a GloMax® Discover microplate reader (Promega, Walldorf, Germany).

Techniques: Control, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Injection, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Complement Activation and STAT4 Expression Are Associated with Early Inflammation in Diabetic Wounds

doi: 10.1371/journal.pone.0170500

Figure Lengend Snippet: (A) C5a concentration in wound beds of diabetic and control mice absorbed by filter paper and assayed by ELISA. Left and right wounds averaged for n = 3 mice in each group and time point: 10 min (P = 0.05) 2h (P = 0.002), 4h (P = 0.001), 24h (P = 0.05). * P ≤0.05 vs. hetero (control). (B) C3-fragment deposition (C3 opsonization) in the subcutaneous tissue at the edges of the wound beds of diabetic and control mice assayed by immunofluorescence (n = 2). (C) Nucleated cell infiltration into the subcutaneous tissue at the edges of the wound beds of diabetic and control mice assayed by DAPI fluorescence (n = 2). Data are means ± SEM.

Article Snippet: The liquid samples were then measured using the mouse Complement C5a ELISA kit (R&D Systems, MN).

Techniques: Concentration Assay, Control, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Fluorescence

Journal: PLoS ONE

Article Title: Complement Activation and STAT4 Expression Are Associated with Early Inflammation in Diabetic Wounds

doi: 10.1371/journal.pone.0170500

Figure Lengend Snippet: (A) C5a concentration in the wound beds of diabetic and heterozygous control mice absorbed by filter paper and assayed by ELISA. Left and right wounds averaged for n = 3 mice in each group and time point: 4H (P = 0.002), 8H (P = 0.007) and 48H (P = 0.05). (B) C5a concentration in the wound beds of diabetic and heterozygous control mice treated with vehicle control gel or PIC1 gel (combined time points). db/db ± PIC1 (P = 0.05). Heterozygous ± PIC1 (P = 0.01); data are means ± SEM. (C) C3-fragment deposition (C3 opsonization) in the subcutaneous tissue at the edges of the wound beds of diabetic and control mice treated with vehicle control gel or PIC1 gel (combined time points). db/db ± PIC1 (P = 0.09). Heterozygous ± PIC1 (P = 0.12), Data are means ± SEM. * P ≤0.05 vs. saline control.

Article Snippet: The liquid samples were then measured using the mouse Complement C5a ELISA kit (R&D Systems, MN).

Techniques: Concentration Assay, Control, Enzyme-linked Immunosorbent Assay, Saline