Cross-sections of carotid arteries were used for confocal microscopic imaging. Platelets were stained with antibodies against CD42d (AF6990, R&D, USA), CD41 (553847, BD, USA) and PF4 (ab282111, Abcam, USA). Monocytes/macrophages were stained with rat anti-mouse F4/80 antibody (MAB5580, R&D, USA). Neutrophils were stained with Ly6G antibody (sc-52515, SANTA CRUZ, USA). Endothelial cells were stained with rabbit anti-mouse CD31 (ab28364, Abcam, USA). Podoplanin expression was determined with Syrian Hamster anti-mouse podoplanin antibody (14-5381-82, eBioscience, USA). Cytoskeletal proteins were determined with phalloidin antibody (CA1610, Solarbio, China). Secondary antibodies Alexa Fluor 488-conjugated donkey anti-rat IgG, Alexa Fluor 488-conjugated goat anti-mouse IgG, Alexa Fluor 647-conjugated donkey anti-rabbit IgG, and Alexa Fluor 647-conjugated goat anti-Syrian hamster IgG were all from Abcam. Donkey anti-sheep IgG NorthernLights™ NL557-conjugated antibody was purchased from R&D. Tissues were counterstained with DAPI before being mounted and examined by confocal microscopy, an Olympus multiphoton laser scanning microscope with high S/N ratio objectives and suppressed autofluorescence, which enables advanced deeper imaging with high resolution (FV1000MPE and FV3000MPE Olympus, Japan).
Multiphoton laser scanning microscope
Manufactured by Olympus
Sourced in Japan
The Multiphoton Laser Scanning Microscope is a high-performance imaging system that utilizes two-photon excitation to capture detailed images of biological samples. It features a tunable, ultrafast pulsed laser source and a scanning system that allows for precise control of the illumination beam.
Automatically generated - may contain errors
Lab products found in correlation
Ca1610, by Solarbio (1 mentions)
Ab28364, by Abcam (1 mentions)
Mab5580, by R&D Systems (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg, by Abcam (1 mentions)
Fv1000mpe, by Olympus (1 mentions)
Ab282111, by Abcam (1 mentions)
Af6990, by R&D Systems (1 mentions)
Alexa fluor 647 conjugated donkey anti rabbit igg, by Abcam (1 mentions)
Freezing microtome, by Leica (1 mentions)
Sc 373717, by Santa Cruz Biotechnology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Fv1200mpe, by Olympus (1 mentions)
5 protocols using multiphoton laser scanning microscope
1
Multicolor Immunostaining of Carotid Artery Sections
2
Quantitative Analysis of Amyloid-β Accumulation
3
Decalcification and Cryosectioning of Bone Samples
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The human and mouse bone samples used for micro-computed tomography scanning were decalci ed in 0.5 M EDTA solution for 4 weeks with the change of solution twice a week. The tissues were then dehydrated using 20% sucrose and 2% polyvinylpyrrolidone solution for 2 week at 4 °C and then embedded in O.C.T. compound (Tissue-Tek). Cryosections (10 μm) were prepared using freezing microtome (Leica, Wetzlar) for immunostaining. The bone sections were washed three times with 0.3% PBST (Triton X-100) and blocked with 5% bovine serum albumin in PBS for 1 hour. Sections were incubated with primary antibody rabbit-anti-human (mouse) Glutathione Peroxidase 4 (GPX4, abcam) at 4 °C overnight. After three washes with 0.3% PBST, the sections were then incubated with uoresceinconjugated secondary antibody together with nuclear counterstaining dye (DAPI) at room temperature in the dark. After another three washes, slides were mounted with 50% glycerol. The processed sections were visualized and imaged using a Multiphoton Laser Scanning Microscope (Olympus).
4
Immunofluorescence Analysis of SMARCA4 Overexpression
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SAS cells transfected with the SMARCA4 overexpression plasmid were plated on glass slides for 24 h. Afterwards, the cell slides were rinsed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 30 min, followed by permeabilization with 0.2% Triton X-100 at room temperature. To avoid non-specific binding, the slides were then incubated with 5% goat serum for 1 h. Primary anti-E-cadherin antibody (1:300, #sc-8426; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and anti-vimentin antibody (1:300, #sc-373717, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were incubated at 4 °C overnight. The cell slides were next incubated with the corresponding secondary antibody (fluorescein isothiocyanate (FITC)-conjugated goat-anti-rabbit for E-cadherin, or DyLight-594-conjugated rabbit-anti-mouse for vimentin) in the dark for 1 h at room temperature. Immunofluorescence images were eventually acquired by confocal laser scanning microscopy using an Olympus Multi-Photon Laser Scanning Microscope (Olympus Corporation, Tokyo, Japan).
5
Organotypic Brain Slice Immunostaining Protocol
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Organotypic brain slices were washed in PBS and then xed on their inserts in 4% paraformaldehyde for 1 h and stained [34] . Individual slice cultures were cut out from their membranes after xation and then treated as free oating sections for the following steps. Slice cultures were permeabilized for 18h in 0.5% Triton X-100 at 4°C and then blocked in 20% BSA (Sigma-Aldrich) for 4 h at room temperature (RT). Slice cultures were then incubated with appropriate primary antibodies overnight at 4°C in 5% BSA, washed and then incubated with uorophore-coupled secondary antibodies for 4h at RT. Slice cultures were washed a nal time before mounting on slides with Fluoromount-G with DAPI (Thermo Fisher Scienti c, P36941) and then imaged by Multiphoton Laser Scanning Microscope (Olympus, FV1200MPE).
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