Inverted sp8 confocal microscope

Manufactured by Leica Microsystems

Sourced in Germany

The Leica Inverted SP8 confocal microscope is a high-performance microscope system designed for advanced imaging applications. It features a confocal detection system that enables high-resolution, optical sectioning of samples. The microscope is capable of capturing detailed images of various specimens, including cells, tissues, and materials.

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Lab products found in correlation

Lsm710nlo two photon microscope, by Zeiss (2 mentions) Lumox dish, by Sarstedt (2 mentions) Glass bottom dish, by Ibidi (2 mentions) Matrigel, by Corning (2 mentions) A22287, by Thermo Fisher Scientific (1 mentions) D3571, by Thermo Fisher Scientific (1 mentions) Leica hc pl apo 1.4 na 63 oil objective, by Leica Microsystems (1 mentions) Inverted sp5 confocal microscope, by Leica Microsystems (1 mentions) Evos xl core imaging system, by Thermo Fisher Scientific (1 mentions) Acti stain 670, by Cytoskeleton (1 mentions) Dragonfly spinning disc confocal microscope, by Oxford Instruments (1 mentions) Dapi fluromount g, by Southern Biotech (1 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions) Power block, by BioGenex (1 mentions)

6 protocols using inverted sp8 confocal microscope

Immunofluorescence Staining Protocol

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Permeabilisation was performed using PBS with 0.1 M glycine, 0.3% Triton X-100 for 30 min at RT. Primary antibodies were incubated overnight at 4°C. All primary antibodies were diluted in blocking buffer (1% BSA, 0.1% Tween in PBS) at 1:200. Cells were then washed three times with 0.1% Tween-PBS before being incubated with fluorescently conjugated Alexa Fluor secondary antibodies diluted 1:500 in 0.1% Tween-PBS for 2 hr at room temperature. Where indicated Alexa Fluor 647 Phalloidin (1:100; A22287, Thermo Fisher Scientific) and DAPI (1:1000; D3571 Thermo Fisher Scientific) were added concomitantly with the secondary antibodies. Primary and secondary antibody lists can be found in Supplementary file 4.
Imaging took place on an inverted SP5 confocal microscope (Leica Microsystems) using a Leica HC PL FLUOTAR 0.5 NA 20.0× dry objective and a Leica HC PL APO 1.4 NA 63× oil objective, or on an inverted SP8 confocal microscope (Leica Microsystems) with a Leica HC PL APO CS2 1.4 NA 63× oil objective and a Leica Fluotar VISIR 0.95 NA 25× water objective. Within each independent experiment, laser power and detector gain were maintained constant.

Mackinlay K.M., Weatherbee B.A., Souza Rosa V., Handford C.E., Hudson G., Coorens T., Pereira L.V., Behjati S., Vallier L., Shahbazi M.N, & Zernicka-Goetz M. (2021). An in vitro stem cell model of human epiblast and yolk sac interaction. eLife, 10, e63930.

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Multicolor Immunofluorescence Imaging

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Alexa Fluor 488 Donkey anti-mouse (1:500; ThermoFisher, A21202), Alexa Fluor 555 Donkey anti-rabbit (1:500; ThermoFisher, A32727), Alexa Fluor 647 Donkey anti-goat (1:500, ThermoFisher, A21447)
F-actin staining: Acti-Stain 670 (1:100; Cytoskeleton, Inc., PHDN1-A)
Immunofluorescence images were acquired on an inverted SP8 confocal microscope (Leica Microsystems) with the 20x/0.75 IMM CS2 (HC PL APO) objective with a z-stack of 2 μm. Image preparation was conducted in Fiji software^{69 (link)}. Laser power and detector gain were maintained constant within a single experiment.
Brightfield images were acquired on an EVOS XL Core Imaging System (AMEX1100, ThermoFisher).

Bergmann S., Penfold C.A., Slatery E., Siriwardena D., Drummer C., Clark S., Strawbridge S.E., Kishimoto K., Vickers A., Tewary M., Kohler T.N., Hollfelder F., Reik W., Sasaki E., Behr R, & Boroviak T.E. (2022). Spatial profiling of early primate gastrulation in utero. Nature, 609(7925), 136-143.

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Imaging Skin Explant Dynamics

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Lateral skin explants were taken from E13.5 embryos (H2B-EGFP^tg/wt) and transferred to media (advanced DMEM + 2 mM L-glutamine + 0.1 mg/ml penicillin/streptomycin + 10% FBS) at 37 °C where they formed rolls. The rolls were embedded in 1% low-melting agarose or Matrigel (Corning) in media at 37 °C, where the cutting edge was touching the membrane-bottom of a Lumox® dish (Sarstedt) or a glass-bottom dish (ibidi). The dishes were covered with media and incubated at 37 °C and 5% CO₂ until imaging. Time-lapse imaging was carried out between E13.5 and E14.5 using an inverted SP8 confocal microscope (Leica microsystems), an inverted Dragonfly Spinning disc confocal microscope (Andor), or an inverted LSM710NLO Two-Photon microscope (Zeiss) with 20× air objective or 40× water or oil immersion objective and incubation at 37 °C and 5% CO₂.

Damen M., Wirtz L., Soroka E., Khatif H., Kukat C., Simons B.D, & Bazzi H. (2021). High proliferation and delamination during skin epidermal stratification. Nature Communications, 12, 3227.

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Imaging Embryonic Skin Explant Dynamics

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Lateral skin explants were taken from E13.5 embryos (H2B-EGFP tg/wt ) and transferred to media (advanced DMEM + 2 mM L-Glutamine + 0.1 mg/ml Penicillin/Streptomycin + 10% FBS) at 37 °C where they formed rolls. The rolls were embedded in 1 % low-melting agarose or Matrigel (Corning) in media at 37 °C, where the cutting edge was touching the membrane-bottom of a Lumox® dish (Sarstedt) or a glass-bottom dish (ibidi). The dishes were covered with media and incubated at 37 °C and 5 % CO2 until imaging. Time-lapse imaging was carried out between E13.5 and E14.5 using an inverted SP8 confocal microscope (Leica microsystems), an inverted Dragonfly Spinning disc confocal microscope (Andor) or an inverted LSM710NLO Two-Photon microscope (Zeiss) with 20x air objective or 40x water or oil immersion objective and incubation at 37 °C and 5 % CO2.

Damen M., Wirtz L., Soroka E., Khatif H., Kukat C., Simons B.D., & Bazzi H. (2020). A two-phase model of skin epidermal stratification: lessons from centrosomes.

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Immunohistochemical Quantification of Dermal Staining

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Immunohistochemistry was performed on 8 μm sectioned paraffin slides which were blocked with 1X Powerblock (HK083‐50 K; Biogenex, Fremont, CA) and incubated for 1 h at 37°C with the relevant primary antibody for 1 h at 37°C. Specimens were washed in phosphate‐buffered saline (10,010,023; Gibco®, Dublin, Ireland) and incubated with a secondary antibody for 1 h at 37°C. Specimens were then washed in phosphate‐buffered saline (10,010,023; Gibco®, Dublin, Ireland) and mounted onto glass slides in DAPI Fluromount‐G (0100–01; SouthernBiotech, Birmingham, AL). Fluorescent images of the dermal layer were taken using an SP8 inverted confocal microscope (Leica Microsystems, Wetzlar, Germany) at 20X magnification (25 images per condition). The percentage of stain‐positive pixels was calculated on each image using ImageJ with a Colour Detect macro to quantify the relevant number of pixels.

Berry C.E., Abbas D.B., Griffin M., Lintel H., Guo J., Kameni L., Churukian A.A., Fazilat A.Z., Chen K., Gurtner G.C., Longaker M.T., Momeni A, & Wan D.C. (2024). Deferoxamine topical cream superior to patch in rescuing radiation‐induced fibrosis of unwounded and wounded skin. Journal of Cellular and Molecular Medicine, 28(8), e18306.

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Scratch Assay for Cell Migration Analysis

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MoMϕs were plated in a monolayer in a 96 wells flat bottom plate and allowed to adhere for 24 hours. Pf SPZ (1:1 ratio), matched equivalent of SGE or Cytochalasin D[51 (link)] (10uM) was added, and plates were spun down at a low centrifugal speed (1000rpm) in order to sediment SPZ. Using a pipet tip a scratch was made in one fluent motion in the monolayer. Plates were incubated at 37°C, 5% CO₂ and imaged after 1 hour and 40 hours using a SP8 inverted confocal microscope (Leica Microsystems, Wetzlar). Images were analyzed using ImageJ 1.48v public domain software (NIH, USA) using the following protocol: Process-> Find edges, process->Sharpen, Image-> Adjust threshold (set threshold manually to reduce background noise), process->Find edges, analyze particles->plot profile. Profile data of scratch area of the image was then loaded into Microsoft Excel and plotted using GraphPad Prism (La Jolla, CA, USA) version 7. The scratch area was located a gray value below 700. In addition, we plotted the difference between the mean gray value over a representative section of the full scratch area per donor comparing T40 with T1.

Winkel B.M., Pelgrom L.R., van Schuijlenburg R., Baalbergen E., Ganesh M.S., Gerritsma H., de Korne C.M., Duszenko N., Langenberg M.C., Chevalley-Maurel S.C., Smits H.H., de Jong E.C., Everts B., Franke-Fayard B, & Roestenberg M. (2020). Plasmodium sporozoites induce regulatory macrophages. PLoS Pathogens, 16(9), e1008799.

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