Sp8 confocal microscope

Manufactured by Olympus

The SP8 confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features state-of-the-art optics, multiple laser excitation sources, and sophisticated detectors to capture high-quality, detailed images of samples.

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Lab products found in correlation

Donkey anti goat cy3, by Jackson ImmunoResearch (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Fv3000, by Olympus (1 mentions) Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions) P1873, by R&D Systems (1 mentions) Col1a1, by Rockland Immunochemicals (1 mentions) Cm1900, by Leica (1 mentions) Alexa fluor 555 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions) Alexa fluor 647 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions) Alexa fluor 555 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions) Hoechst solution, by Thermo Fisher Scientific (1 mentions) Ix71 fluorescence microscope, by Olympus (1 mentions) Fluoview 3000rs confocal microscope, by Olympus (1 mentions) Prism 8, by GraphPad (1 mentions) Imagej, by GraphPad (1 mentions) Fluoview fv3000 confocal microscope, by Olympus (1 mentions) Spe confocal microscope, by Leica (1 mentions) Two photon microscope, by Olympus (1 mentions)

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Confocal microscope (790 citations) TCS SP8 confocal microscope (6 citations)

8 protocols using sp8 confocal microscope

Decalcified Tissue Immunohistochemistry and Staining

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Tissues were fixed in 4% paraformaldehyde for 48 h, incubated in 15% DEPC-EDTA (pH 7.8) and ultrasonically decalcified. The specimens were embedded in paraffin or OCT and cut into 7 μm sections.
Immunofluorescence assay: Sections were blocked in PBS with 10% horse serum and 0.1% Triton for 1 h at room temperature. Then, the cells were stained overnight with rabbit anti-perilipin A/B (Sigma, P1873, 1:1 000, USA) and OPN (1:1 000; R&D, AF808). Donkey-anti-rabbit Alexa Fluor 488 (1:1 000; Molecular Probes, A21206) and donkey-anti-goat Cy3 (1:1 000; Jackson ImmunoResearch, 705–165–147) were used as secondary antibodies. DAPI (Sigma, D8417) was used for counterstaining. Slides were mounted with anti-fluorescence mounting medium (Dako, S3023), and images were acquired with an Olympus FV3000 and SP8 confocal microscope.
Immunohistochemical staining and Col1a1 (1:100; Rockland, 600–400–103) staining were performed as described by Dako.
Tissue sections were used for TRAP, BODIPY, and Oil Red O staining according to the standard protocol.

Suo J., Zou S., Wang J., Han Y., Zhang L., Lv C., Jiang B., Ren Q., Chen L., Yang L., Ji P., Zheng X., Hu P, & Zou W. (2022). The RNA-binding protein Musashi2 governs osteoblast-adipocyte lineage commitment by suppressing PPARγ signaling. Bone Research, 10, 31.

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Spinal Cord Immunohistochemistry of Ion Channels

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Rats were intracardially perfused with ice-cold 4% paraformaldehyde (in 1× PBS)
under deep anesthesia. Spinal cord were postfixed for 4 hours at 4 °C, then
transfer to 0.1 M PB solution containing 30% (w/v) sucrose for dehydro before
sectioning (20 µm; Leica CM1900). Spinal cord slices were permeabilized with
0.2% Triton X-100 in PBS at room temperature and then blocked in 3% goat serum
and 5% bovine serum albumin (BSA) with 0.05% Triton X-100 in PBS for 1 hour.
Then incubated in rabbit anti-BKα (AP107, Alomone lab, 1:500) and Guinea pig
anti-NMDAR1 (GluN1) (1:500) in 5% BSA at 4 °C for 48 h. Slices were washed with
PBS and then incubated in Alexa 488 labeled donkey anti-rabbit serum (1:1000)
and Alexa 594 labeled goat anti-guinea pig (1:1000) in PBS for 2 hours and then
washed again. All sections were stained with DAPI, mounted with antifade
solution, and imaged with an Olympus SP8 confocal microscope.

Fan F., Chen Y., Chen Z., Guan L., Ye Z., Tang Y., Chen A, & Lin C. (2021). Blockade of BK channels attenuates chronic visceral hypersensitivity in an IBS-like rat model. Molecular Pain, 17, 17448069211040364.

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Quantifying Anterograde Axonal Tracing

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Fluorescence z stack images were acquired with a Leica SP8 confocal microscope and an Olympus FV1000 confocal microscope, and mosaic images were stitched using the TileScan function of LAS X software. Images were minimally processed with Fiji/ImageJ software (NIH) to enhance brightness and contrast. Median filters were used to decrease noise. For co-localization, analysis quantification was done in single confocal z sections. Cell counting was performed using the ImageJ Cell Counter plug-in. Quantification of anterograde genetically labeled PSDC axons was done as described (Grider et al., 2006 (link)). Briefly, the Hessian filter of Feature J was applied, selecting for the smallest eigen values. The resulting eigen image was converted into a binary image by applying a threshold in ImageJ. The number of pixels corresponding to positive mCherry labeling was summed for each ROI (gracile and cuneate nuclei), and the degree of innervation was expressed as a percentage of positive labeling in the areas. Finally the values obtained from the 3 anterior-posterior regions analyzed were averaged for each mouse.

Paixão S., Loschek L., Gaitanos L., Morales P.A., Goulding M, & Klein R. (2019). Identification of Spinal Neurons Contributing to the Dorsal Column Projection Mediating Fine Touch and Corrective Motor Movements. Neuron, 104(4), 749-764.e6.

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Immunofluorescence Staining of Cells

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Cells were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100, followed by blocking with 10% FBS in PBS for 1 h. Samples were stained with primary antibodies for overnight at 4 °C. Secondary antibody staining was performed for 1 h at room temperature with Alexa Fluor 488-donkey anti-rabbit IgG, Alexa Fluor 555-donkey anti-goat IgG, Alexa Fluor 647-donkey anti-mouse IgG, Alexa Fluor 488-donkey anti-goat IgG and Alexa Fluor 555-donkey anti-mouse IgG (ThermoFisher Scientific). Hoechst staining was performed for 2 minutes at room temperature with Hoechst solution (ThermoFisher scientific, 62249). Images were taken using Leica SP8 con-focal microscope or Olympus IX71 fluorescence microscope.

Han D., Liu G., Oh Y., Oh S., Yang S., Mandjikian L., Rani N., Almeida M.C., Kosik K.S, & Jang J. (2023). ZBTB12 is a molecular barrier to dedifferentiation in human pluripotent stem cells. Nature Communications, 14, 632.

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Multimodal Microscopy for Tissue Imaging

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Except for ChAT microscopy, all other imaging was done by using the oil immersion 63 X objective on the Leica SP8 confocal microscope and by using the oil immersion 40 X objective on the Olympus Fluoview 3000rs confocal microscope with resonance scanning mode. For thick tissues, such as human tissues, the Galvano mode of the Olympus Fluoview 3000rs microscope that enabled higher resolution imaging and averaging was used. Images obtained were then analyzed using Fiji (https://fiji.sc/).
Live tissue imaging of the mouse gut tissue was performed by harvesting small intestinal tissue from an adult Wnt1-Cre:Rosa26^lsl-tdTomato mouse and immediately putting it in OptiMEM solution. The tissue was then immediately put on a chamber slide containing OptiMEM and imaged in live tissue culture conditions of the EVOS M7000 microscope under the 20 X objective.

Kulkarni S., Saha M., Slosberg J., Singh A., Nagaraj S., Becker L., Zhang C., Bukowski A., Wang Z., Liu G., Leser J.M., Kumar M., Bakhshi S., Anderson M.J., Lewandoski M., Vincent E., Goff L.A, & Pasricha P.J. (2023). Age-associated changes in lineage composition of the enteric nervous system regulate gut health and disease. eLife, 12, RP88051.

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Quantifying Eye-Antennal Disc Clones

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Eye-antennal disc images were taken with a Leica SP8 confocal microscope or Olympus Fluoview FV3000 confocal microscope. To measure clone size, ImageJ (Fiji) software was used to determine the threshold of the fluorescence. Total clone area/disc area (%) in the eye-antennal disc was calculated using ImageJ and Prism 8 (GraphPad).

Wang Z., Xia X., Li J, & Igaki T. (2022). Tumor elimination by clustered microRNAs miR-306 and miR-79 via noncanonical activation of JNK signaling. eLife, 11, e77340.

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Visualizing T Cell Tumor Infiltration

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The hybrid tumor-CAF spheroids were formed using the fabricated devices. The on-chip T cell infiltration of tumor spheroids was tracked and recorded by a Leica SP8 confocal microscope or Olympus OSR spinning disk confocal microscope, and raw images captured were exported as TIFF image stacks in ImageJ. The detailed on-chip investigation of T cell tumor infiltration as described in the SI Appendix, SI Materials and Methods.

Ao Z., Cai H., Wu Z., Hu L., Nunez A., Zhou Z., Liu H., Bondesson M., Lu X., Lu X., Dao M, & Guo F. (2022). Microfluidics guided by deep learning for cancer immunotherapy screening. Proceedings of the National Academy of Sciences of the United States of America, 119(46), e2214569119.

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Multimodal Imaging and Analysis Techniques

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IF images were acquired using a Leica SPE confocal microscope, a Leica SP8 confocal microscope or an in house-built Olympus two-photon microscope (Multiphoton Microcopy Core, Massachusetts General Hospital), depending on the experiment. IHC and ISH images were acquired with a Olympus DP72 microscope. Metabolites in the colorectal adenoma were imaged using a timsTOF fleX mass spectrometer (Bruker Daltonics, Billerica, MA). Graphpad Prism 6 and Microsoft Excel v16.15 were used for statistical analyses throughout this study. ImageJ 1.51 u was used to process IF images. STAR was used to map RNA-sequencing data. HTseq was used to assign reads to Gencode vM9. Pathway enrichment analysis was carried out using GSEA as detailed in the experimental procedures. MSI data were visualized using SCiLS Lab software (version 2021c premium, Bruker Daltonics, Billerica, MA). Flow cytometry data was analyzed by FlowJo vX.0.7.

Sebastian C., Ferrer C., Serra M., Choi J.E., Ducano N., Mira A., Shah M.S., Stopka S.A., Perciaccante A.J., Isella C., Moya-Rull D., Vara-Messler M., Giordano S., Maldi E., Desai N., Capen D.E., Medico E., Cetinbas M., Sadreyev R.I., Brown D., Rivera M.N., Sapino A., Breault D.T., Agar N.Y, & Mostoslavsky R. (2022). A non-dividing cell population with high pyruvate dehydrogenase kinase activity regulates metabolic heterogeneity and tumorigenesis in the intestine. Nature Communications, 13, 1503.

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