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: 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

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