Xenograft tumor tissues were dissected and embedded in optimal cutting temperature compound (OCT compound) at −20°C. Tissue section slides were obtained by sectioning frozen tissues with CryoStar NX50 (Epredia, MI). OCT compound was removed from section slides with Phosphate-Buffered Saline with 0.1% Tween 20 Detergent (PBST) for 10 min. Section slides were blocked with 1% BSA for 30 min at room temperature and then incubated at 4°C overnight with primary antibodies: rabbit monoclonal F4/80 antibody (1:100, ThermoFisher Scientific, Cat# MA5–16363, RRID:AB_2537882), rat monoclonal CD90.2 (Thy-1.2) (1:100, ThermoFisher Scientific, Cat# 14–0902-82, RRID:AB_467379), mouse monoclonal phospho-Axl (Tyr779) (1:100, ThermoFisher Scientific, Cat# MA5–24334, RRID:AB_2609001), and rabbit polyclonal IL-11 (1:200, ThermoFisher Scientific, Cat# PA595982, RRID:AB_2807784). Tissue section slides were then incubated with secondary antibodies for 1 h under room temperature and mounted by mounting medium with DAPI (Sigma Aldrich, DUO82040–5ML). Images were captured by Zeiss LSM 710 confocal microscope and exported by ZEN blue software (Zeiss).
Cryostar nx50
Manufactured by Epredia
The CryoStar NX50 is a cryogenic microtome designed for sectioning frozen tissue samples. It features a motorized cutting mechanism and a temperature-controlled cryochamber to maintain the desired cutting temperature.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Green fluorescent beads, by Thermo Fisher Scientific (2 mentions)
Zen blue software, by Zeiss (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
A1 confocal imaging system, by Nikon (1 mentions)
Nbp1 92693, by Novus Biologicals (1 mentions)
5 protocols using cryostar nx50
1
Immunofluorescence Staining of Xenograft Tumors
2
Fluorescent Bead Injection in Mouse Brain
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This study was performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and the University of Maryland, School of Medicine, Animal Care and Use Committee. One male C57BL6J mouse was used for in vivo testing. One microliter of green fluorescent beads (505/515, 0.1 μm diameter, Thermofisher, #F8803, 25 times diluted with phosphate buffer solution (PBS) was injected into the cerebral cortex using either pulled glass pipettes (right side of the hemisphere) connected to a Narishige micromanipulator (#MO10) or Hamilton syringe needle 33 Gauge (left side of the brain) connected to a motorized pump (KD Scientific, #78–8130). The Bregma coordinates for the three injection sites for each hemisphere were −1, 0, and 1 mm. Midline and depth were the same among the sites: 2 and 0.8 mm, respectively. The injection speed was 0.5 μL/min. The animal was perfused with 4% paraformaldehyde 11 days after the injection of fluorescent beads and dehydrated in 30% sucrose in PBS before being cryosectioned (Cryostar NX50, Epredia) at 40 μm for observation under fluorescence microscopy (Leica).
3
Fluorescent Bead Injection in Mouse Brain
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This study was performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and the University of Maryland, School of Medicine, Animal Care and Use Committee. One male C57BL6J mouse was used for in vivo testing. One microliter of green fluorescent beads (505/515, 0.1 μm diameter, Thermofisher, #F8803, 25 times diluted with phosphate buffer solution (PBS) was injected into the cerebral cortex using either pulled glass pipettes (right side of the hemisphere) connected to a Narishige micromanipulator (#MO10) or Hamilton syringe needle 33 Gauge (left side of the brain) connected to a motorized pump (KD Scientific, #78–8130). The Bregma coordinates for the three injection sites for each hemisphere were −1, 0, and 1 mm. Midline and depth were the same among the sites: 2 and 0.8 mm, respectively. The injection speed was 0.5 μL/min. The animal was perfused with 4% paraformaldehyde 11 days after the injection of fluorescent beads and dehydrated in 30% sucrose in PBS before being cryosectioned (Cryostar NX50, Epredia) at 40 μm for observation under fluorescence microscopy (Leica).
4
Dual Immunofluorescence Staining of Lung Cells
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Lung tissue was sliced into 20 μm sections using a CryoStar NX50 (Epredia, Portsmouth, NH). Slides were then fixed in PFA 4% and blocked using 10% goat serum in 0.2% Triton X-100/PBS 1X. Anti-AQP5 (Novus Biotechnology) and anti-SFTPC (EpigenTek, Farmingdale, NY) were used at dilution 1:1000 for AEC1 and AEC2 staining, respectively. Lung fibroblasts were stained with FSP-1 antibody (MilliporeSigma) at a dilution of 1:750. Nuclei were stained with DAPI. Imaging was performed with a Nikon A1 Confocal Imaging System.
5
Immunohistochemistry of Iba1 and NeuN in Ischemic Stroke
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On Day 14, animals were transcardially perfused using ice cold phosphate buffered saline and 4% paraformaldehyde (pH 7.4). Brains were harvested and soaked in 4% paraformaldehyde for 24 hours before being transferred to 30% sucrose prior to sectioning in 30 μm slices using a cryostat (Epredia CryoStar NX50). The primary antibodies were rabbit polyclonal ionized calcium-binding adapter molecule 1 (Iba1, 1:1500, FujiFilm Wako Pure Chemical Corporation catalog #019-19741) and mouse monoclonal RBFOX3/NeuN (1:1000, Novus Biologicals catalog #NBP1-92693). Secondary antibodies used were goat-anti-rabbit Alexa Fluor 488 (1:500, Thermo Fisher Scientific, catalog #A11070) and goat-anti-mouse Alexa Fluor 568 (1:500, Thermo Fisher Scientific, catalog #A11004). Sections were imaged using laser scanning confocal microscopy (Nikon C2+) using 405-, 488-, and 561-nm lasers for excitation of DAPI, Alexa 488, and Alexa 568, respectively. Images were taken in the penumbral region of the ipsilateral cortex to the MCAO (n = 3 images/animal) before being counted using ImageJ.
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