At the time of sacrifice, hearts were perfused with GFP-conjugated Isolectin B4 from Griffonia simplicifolia (Vector Lab) for 15 min at room temperature. Hearts were then fixed in 4% paraformaldehyde overnight before embedding in OCT compound (Thermo Scientific) with dry ice. In all, 10- µm cross-sections of the heart were made by using HM525 NX Cryostat (Thermo Scientific) starting from the apex to top. The sections were stored in −80 oC before use. The number of capillaries were counted in five random microscopic fields using a fluorescence microscope (Nikon) and expressed as the number of capillaries per square millimeter tissue.
Hm525 nx cryostat
Manufactured by Thermo Fisher Scientific
Sourced in United States
The HM525 NX Cryostat is a laboratory instrument used for the preparation of thin tissue sections for microscopic analysis. It is designed to maintain a controlled low-temperature environment to facilitate the sectioning of frozen tissue samples.
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Lab products found in correlation
Oct compound, by Thermo Fisher Scientific (4 mentions)
Surgipath, by Leica Microsystems (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Axioplan 2 fluorescence microscope, by Zeiss (2 mentions)
Polylysin coated glass slides, by Thermo Fisher Scientific (2 mentions)
Anti human nuclei antibody, by Merck Group (2 mentions)
Alexa fluor 488 conjugated goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (2 mentions)
Vectashield containing dapi, by Vector Laboratories (2 mentions)
Oct compound, by Sakura Finetek (2 mentions)
Senescence detection kit, by Abcam (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Vs120 virtual slide microscope, by Olympus (1 mentions)
Sc 1050, by Santa Cruz Biotechnology (1 mentions)
Frozen section compound, by Leica camera (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Geltrex, by Thermo Fisher Scientific (1 mentions)
Essential 8 basal medium, by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
17 protocols using hm525 nx cryostat
1
Capillary Quantification in Cardiac Tissue
2
Cryogenic Tissue Sectioning and AFM
3
Immunohistochemical Analysis of Corneal Samples
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For immunohistochemistry, excised corneal samples were embedded in frozen section compounds (Surgipath; Leica Microsystems, Nussloch, Germany), and stored at −80 °C until sectioning. Serial sections of 10 µm sections were cut using a HM525 NX cryostat (Thermo Scientific) and collected on polylysin-coated glass slides (Thermo Scientific). Samples were rinsed and blocked in 5% normal goat serum in PBS for 30 min at room temperature. Subsequently, samples were incubated with the primary antibodies at room temperature for 1 hour or at 4 °C overnight. The primary antibodies used were Na+/K+-ATPase and ZO-1, as well as anti-human nuclei antibodies (Merck Millipore, Massachusetts, USA). Subsequently, samples were labeled with an AlexaFluor 488 conjugated goat anti-mouse IgG secondary antibody (2.5 µg/ml, Life Technology), mounted in Vectashield containing DAPI (Vector Laboratories, California, USA), and visualized using a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany).
4
Transcardial Brain Perfusion and Sectioning
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A subset of mice was anesthetized and transcardially perfused with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde (PFA) (Sigma, St. Louis, MO) in PBS. Fixed brains were extracted and incubated overnight in 4% PFA on a rocker at 4°C. Brains were rinsed three times with PBS for 30 min before being submerged overnight in 30% sucrose with 0.03% sodium azide on a rocker at 4°C. The sucrose solution was replaced and left at 4°C for at least 3 days. Brains were embedded in optimal cutting temperature (O.C.T.) compound (Sakura Finetek, Torrance, CA), sectioned into 30-μm slices on a Thermo Scientific HM525NX cryostat (Waltham, MA), and placed on glass microscope slides. Fluorescence microscopy was performed on a Zeiss microscope (Thornwood, NY), and images were captured with Zeiss Zen software.
5
Senescence Detection in Murine Lungs
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The right middle lung lobe of additional animals was inflated with Tissue-Tek O.C.T. Compound (#4583, Sakura Finetek USA, Inc., Torrance, CA, United States) through the trachea, embedded in Tissue-Tek O.C.T. Compound and frozen in a bath of isopentane pre-cooled in liquid nitrogen. Five μm cryosections were cut using a cryostat (HM525 NX cryostat, Thermo Fisher Scientific, Waltham, MA, United States), and tissue was processed with the Senescence Detection Kit (#K320-250, BioVision Inc., Milpitas, CA, United States), following the manufacturer’s protocol. Briefly, sections were fixed 10 min with the Fixative Solution of the kit at RT, and then incubated overnight at 37°C with the Staining Solution Mix containing 1 mg/ml X-gal. Development of the blue color linked with the beta-galactosidase activity was followed under the microscope. N = 3 animals/genotype.
6
Capillary Density Quantification in Cardiac Tissue
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At the time of sacrifice, hearts were perfused with rhodamine-conjugated isolectin B4 from Griffonia simplicifolia for 15 min at room temperature. After that, hearts were fixed in 4% paraformaldehyde overnight before embedding in an OCT compound (Thermo Scientific) with dry ice. Transverse cryosection (10 µm) of the fixed tissue was performed using the HM525 NX Cryostat (Thermo Scientific) from the apex to the superior border. These sections were stored in −80 °C before use. The number of capillaries was counted in five random microscopic fields using a fluorescence microscope (Nikon) and expressed as the number of capillaries per square millimeter tissue.
7
Cryostat Sectioning of Frozen Skeletal Muscle
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Skeletal muscle samples frozen in OCT were cut using an HM525NX cryostat (Thermo Fisher) at -24 °C and 8 μm sections were placed on charged slides and dried for one hour. Slides were then used for histochemical analysis (described below) or stored at -80 °C for future experiments.
8
Quantifying Pancreatic Islet Inflammation
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Frozen pancreases were sectioned (4–5 μm thickness; HM525NX Cryostat, Thermo Fisher Scientific, Scoresby, VIC, Australia), fixed with 100% ethanol at room temperature for 10 min and allowed to dry for 15 min at room temperature. Sections were stained using H&E, imaged (×40 dry objective, VS120 Virtual Slide Microscope; Olympus Australia, Notting Hill, VIC, Australia) and quantified for insulitis and insulitis index [8 ] (OlyVIA software version 3.7, Olympus Australia). A range of 22–37 islets, spaced over 25 μm apart, over four serial sections were imaged per mouse. Standard immunofluorescence staining methods previously described [9 (link)] were used to assess the levels of ER stress (glucose-related protein 78 [GRP78] antibody; sc-1050; Santa Cruz Biotechnology, TX, USA; 1:25), macrophages (F4/80 antibody; CI:A3-1; Abcam, Cambridge, UK; 1:200), neutrophil infiltration (Ly6G6c antibody; NIMP-R147, Abcam; 1:200) and STAT3 phosphorylation (STAT3p antibody; D3A7, Cell Signaling, Danvers, MA, USA; 1:500). The area in pixels/mm2 was quantified using 3–4 islets per mouse using Image J software version 1.45 s.
9
Cerebellar Organoid Electroporation Protocol
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Human iPSCs (ATCC-DYS0100) were maintained in self renewal on a layer of Geltrex (Gibco, A14133-01), in Essential 8 Basal Medium (Gibco, A15169-01) supplemented with E8 Supplement (50X, Gibco A15171-01) and penicillin (100 U/ml)/streptomycin (100 μg/ml) (Gibco, 15140-122). All cells were mycoplasma free. iPSCs were dissociated with 0.5 mM EDTA (pH 8.0; Invitrogen, 15575-038) for 3-min incubation to maintain cell clusters. Cerebellar organoids were cultured as previously described (31 (link), 55 (link)) and were electroporated at 35 days of differentiation protocol with pPBase, pPB CAG LSL Venus, pPB CAG LSL Gfi1:FLAG-IRES-GFP, pPB CAG LSL MYC, and either pS100b-cre or pSox2-cre resuspended in buffer 5 (under patent) (9 (link)). For the 24-hour analysis, organoids were electroporated at 35 days of differentiation protocol with pPB CAG LSL Venus and either pS100b-cre or pSox2-cre. Organoids were transferred inside the electroporation cuvettes (2 mm; VWR, ECN 732-1136), and electroporation was performed with the Gene Pulser XcellTM. Twenty-four hours after electroporation or 39 days after electroporation, they were fixed in 4% PFA, cryoprotected in 30% sucrose, and embedded in Frozen Section Compound (Leica, 3801480). Organoids were cryosectioned at 40 μm with Thermo Fisher Scientific HM525 NX cryostat.
10
Microglial Quantification in Hypothalamus
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Fixed brains were sectioned into 30-μm free-floating slices on a ThermoScientific HM525NX cryostat (Waltham, MA), and subjected to citrate-based antigen retrieval followed by incubation with rabbit anti-Iba1 (FUJIFILM Wako Chemicals USA, Cat. No.019-19741, Richmond, VA, 1:1000) overnight at 4°C. The sections were visualized with DAB and counterstained with hematoxylin. Microscopy was performed on an Olympus BX43 light microscope (Olympus Corporation, Center Valley, PA). For each animal, 2 sections were sampled between -1.0 and -1.8 mm from bregma, and 6 medial hypothalamus fields were systematically captured for each section. Manual cell counts were averaged across 12 fields for each animal.
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