Following 7 days of morroniside treatment, rats were anesthetized intravenously (30 mg/kg 5% pentobarbital sodium; Sigma Aldrich; Merck KGaA, Darmstadt, Germany) and brains were harvested. Brain tissues were mounted in optimal cutting temperature medium (Sakura Finetek USA, Inc., Torrance, CA, USA), frozen at −20°C in a Leica cryostat device (Leica Microsystems GmbH, Wetzlar, Germany) and cut into 4-µm sections. Sections were treated with 3% H2O2 solutions for 20 min at 37°C, blocked with normal goat serum (Sigma Aldrich; Merck KGaA) for 30 min at 37°C, and incubated with anti-MMP2 (cat. no. sc-13594) and anti-MMP9 (cat. no. sc-21733; 1:200; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) antibodies at 4°C for 20 h. Sections were subsequently incubated with Cy3®-labelled goat anti-mouse immunoglobulin (Ig)G (cat. no. ab97035; 1:100; Abcam, Cambridge, MA, USA) in the dark at 37°C for 2 h. DAPI solution was used for nuclear staining at 37°C for 10 min. Slides were observed using a fluorescence microscope (magnification, ×200) and assessed using Image-Pro Plus 6 software (Media Cybernetics, Inc., Rockville, MD USA).
Optimal cutting temperature medium
Manufactured by Sakura Finetek
Sourced in United States, Japan
Optimal cutting temperature medium is a product used for embedding and preserving tissue samples for cryosectioning. It is a non-toxic, water-soluble medium that provides support and protection for the tissue during the freezing process, enabling thin sections to be cut without damage.
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Lab products found in correlation
Dihydroethidium staining, by Thermo Fisher Scientific (2 mentions)
Bx53 microscope, by Olympus (2 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Anti mmp 2, by Merck Group (1 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
A1r inverted confocal microscope, by Nikon (1 mentions)
Oil red, by Merck Group (1 mentions)
Bz x710 microscope, by Keyence (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Mildform 10nm, by Fujifilm (1 mentions)
Midazolam, by Meiji Seika Pharma (1 mentions)
Butorphanol, by Meiji Seika Pharma (1 mentions)
Wild type c57bl 6j mice, by Jackson ImmunoResearch (1 mentions)
Cm3050, by Leica (1 mentions)
Isoflurane, by Abbott (1 mentions)
Mhc antibody, by Developmental Studies Hybridoma Bank (1 mentions)
Pax7 antibody, by Santa Cruz Biotechnology (1 mentions)
Bx61vs fluorescence microscope, by Olympus (1 mentions)
710 laser scanning confocal microscope, by Zeiss (1 mentions)
30 protocols using optimal cutting temperature medium
1
Evaluating MMP2 and MMP9 Expression in Rat Brains
2
Quantitative RNA Expression Profiling in Mouse Eyes
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Enucleated mouse eyes were embedded in optimal cutting temperature medium (Sakura Finetek USA, Torrance, CA, USA) and snap-frozen in liquid nitrogen-supercooled isopentane. Seven-micrometer–thick sections were hybridized with RNAscope probes for Ccl2 (ID: 311791), Il6 (ID: 315891), and Vegfa (ID: 412261) according to manufacturer's instructions (ACDBio, Newark, CA, USA). Sections were mounted in Invitrogen ProLong Gold Antifade Mountant with DAPI (Thermo Scientific, Waltham, MA, USA) and imaged on a Nikon A1R inverted confocal microscope (Nikon Instruments Inc., Melville, NY, USA). Quantification of absolute transcripts was performed in ImageJ. The integrated density of an individual punctum was measured as the first peak in the intensity histogram of each 8-bit grayscale image, thresholded to reduce background. Then, the following equation was used to calculate the total number of transcripts: .
3
Histological Evaluation of NAFLD
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Liver tissue was fixed with formalin and embedded in paraffin, which was ready for section. Haematoxylin and eosin (H&E) staining was performed on liver sections (4 μm thick) with commercially available kit (Nanjing Jiancheng Institute of Bio Engineering, Inc., Nanjing, China)) to visualize the histology. The NAFLD activity scoring (NAS) system including steatosis, lobular inflammation and hepatocellular ballooning (Supplementary Table 1 )12 (link) was employed to evaluate the severity of liver disease. The histology scoring was performed by three pathologists independently.
Oil red (Sigma, MO, USA) staining was performed on the liver tissue embedded in Optimal Cutting Temperature medium (Sakura Finetek, Torrance, CA) to visualize the lipid droplets in the hepatocytes.
Oil red (Sigma, MO, USA) staining was performed on the liver tissue embedded in Optimal Cutting Temperature medium (Sakura Finetek, Torrance, CA) to visualize the lipid droplets in the hepatocytes.
4
Assessing Aortic Superoxide Levels
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Superoxide level in the aortic rings was assessed with dihydroethidium staining (Thermo Fisher Scientific, Waltham, MA), as previously described.25 Aortic rings were snap‐frozen with liquid nitrogen and embedded in optimal cutting temperature medium (Sakura Finetek Japan, Tokyo, Japan). The frozen samples were cut immediately into 10‐μm‐thick sections and mounted on glass slides. The samples were incubated at room temperature for 30 minutes with 2.0×10−6 mol/L dihydroethidium and protected from light. Images were obtained with a BZ‐X710 microscope (Keyence, Osaka, Japan) with an excitation wavelength of 540 nm and an emission wavelength of 605 nm. The fluorescence intensity of dihydroethidium staining was measured semiquantitatively using software (Keyence).
5
Immunofluorescence Staining of Testis Sections
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For immunofluorescence staining, testes fixed with 4% paraformaldehyde were cryo-embedded in an optimal cutting temperature medium (Sakura Finetek, Torrance, CA, USA) and sectioned at a thickness of 4 μm. The frozen testis sections were blocked by incubation in 5% normal serum and 0.1% Triton X-100 (Hyclone, Logan, Utah, USA) in PBS for 1 h at room temperature, followed by incubation with primary antibody overnight at 4°C. The next day, the sections were incubated with secondary antibody for 30 min followed by DAPI (blue) staining of nuclei. The primary and secondary antibodies used in this process are listed in Table 1
6
Euthanasia and Tissue Fixation of Common Marmosets
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Common marmosets were euthanized by exsanguination under deep anesthesia with a mixture of medetomizine (120 μg/kg), midazolam (600 μg/kg) and butorphanol (600 μg/kg; Meiji Seika Pharma Co., Ltd., Tokyo, Japan). Specimens of the GI tract were removed, washed with ice-cold PBS and fixed overnight with Mildform 10NM (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Subsequently, tissue samples were soaked in 30% sucrose in PBS overnight at 40°C and frozen in Optimal Cutting Temperature medium (Sakura Finetek, Tokyo, Japan) at -80°C.
7
Tissue Distribution of FMDV in Cattle
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For the objective of evaluating tissue distribution of FMDV during early infection, two nonvaccinated cattle were euthanized for tissue harvest at 6, 12, 48, and 72 hpe, and four animals were euthanized at 24 hpe. For evaluation of tissue distribution during the persistent phase of infection, six vaccinated and six nonvaccinated cattle were euthanized at 35 dpe. A standardized necropsy procedure with collection of 18 to 22 distinct tissue samples (Tables 1 and 2 ) was performed immediately after euthanasia. Each tissue sample was divided into 30-mg aliquots, which were placed in individual tubes before immediately being frozen over liquid nitrogen. An adjacent specimen from each tissue was divided into two or four replicates, embedded in optimal cutting temperature medium (Sakura Finetek, Torrance, CA) in cryomolds, and frozen over liquid nitrogen. Tissue samples were kept frozen in the vapor phase over liquid nitrogen and were transferred to the lab within 2 h after collection for storage at −70°C until further processing.
8
Retinal Cryosectioning and Immunostaining
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These studies were conducted under protocols approved by the University of California at Los Angeles (UCLA) Animal Research Committee. All experiments were carried out in accordance with the guidelines for the welfare of experimental animals issued by the U.S. Public Health Service Policy on Human Care and Use of Laboratory Animals and the University of California, Los Angeles (UCLA) Animal Research Committee. Wild-type C57BL/6J mice (20–30 g; Jackson Laboratory, Bar Harbor, ME, USA) of both sexes were used for these studies. Animals were 2–3 months old at the time of the experiments. Animals were deeply anesthetized with 1%–3% isoflurane (Abbott Laboratories, North Chicago, IL, USA) and euthanized by cervical dislocation. To prepare vertical cryostat sections of the retina, the eyecups were fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), pH 7.4, for 15–60 min at room temperature (RT). Eyecups were then transferred to 30% sucrose in PB overnight at 4°C. The eyecups were embedded in optimal cutting temperature medium (Sakura Finetek, Torrance, CA, USA) and sectioned at 12–14 μm with a Leica CM3050S (Leica Microsystems, Buffalo Grove, IL, USA). Tissue sections were mounted onto gelatin-coated slides and sections were stored at −20°C until immunostaining.
9
Histological Analysis of Skeletal Muscle
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Extensor digitorum longus and soleus muscles for histology were frozen in isopentane cooled in liquid nitrogen and mounted in optimal cutting temperature medium (Sakura Finetek). The staining for satellite cell (SC; Pax7) and the fibre typing of muscle was determined by immunofluorescence using respectively Pax‐7 antibody (Santa Cruz; 1:40) and mixtures of the following monoclonal anti‐myosin heavy chain (MHC) antibodies (Developmental Studies Hybridoma Bank, University of Iowa): BA‐F8 (IgG2b, 1:100 dilution) specific for MHC‐I, SC‐71 (IgG1, 1:100 dilution) for MHC‐IIA and BF‐F3 (IgM, 1:100 dilution) for MHC‐IIB. Type‐IIX fibres have not been marked and appear black. The skeletal muscle membrane was stained by immunofluorescence for dystrophin (Abcam; 1:200 dilution), and the nuclei were marked with DAPI. Cryosections of the EDL and soleus were also stained for succinate dehydrogenase (SDH). The images were collected with the Olympus BX61VS fluorescence microscope. Cross‐sectional area (CSA) and the number of SC and nuclei of more than 500 fibres stained for MHC (×20 magnification) and 200 fibres stained for SDH per muscle were measured using ImageJ software (Fiji is Just; Version 1.52p; National Institutes of Health, USA). The total number of fibres and SCs was counted in the whole EDL and soleus muscles.
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Extensor digitorum longus and soleus muscles for histology were frozen in isopentane cooled in liquid nitrogen and mounted in optimal cutting temperature medium (Sakura Finetek). The staining for satellite cell (SC; Pax7) and the fibre typing of muscle was determined by immunofluorescence using respectively Pax‐7 antibody (Santa Cruz; 1:40) and mixtures of the following monoclonal anti‐myosin heavy chain (MHC) antibodies (Developmental Studies Hybridoma Bank, University of Iowa): BA‐F8 (IgG2b, 1:100 dilution) specific for MHC‐I, SC‐71 (IgG1, 1:100 dilution) for MHC‐IIA and BF‐F3 (IgM, 1:100 dilution) for MHC‐IIB. Type‐IIX fibres have not been marked and appear black. The skeletal muscle membrane was stained by immunofluorescence for dystrophin (Abcam; 1:200 dilution), and the nuclei were marked with DAPI. Cryosections of the EDL and soleus were also stained for succinate dehydrogenase (SDH). The images were collected with the Olympus BX61VS fluorescence microscope. Cross‐sectional area (CSA) and the number of SC and nuclei of more than 500 fibres stained for MHC (×20 magnification) and 200 fibres stained for SDH per muscle were measured using ImageJ software (Fiji is Just; Version 1.52p; National Institutes of Health, USA). The total number of fibres and SCs was counted in the whole EDL and soleus muscles.
10
Fluorescent Imaging of Muscle Fiber Cross-Section
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For fluorescent imaging, dissected TA muscles were positioned on glass coverslips and mounted on cork disks using optimal cutting temperature medium (Sakura Finetek, Torrance, CA USA) before snap freezing in liquid-nitrogen-cooled isopentane. The glass coverslip was removed and 10-μ transverse sections were cryosectioned, air dried, and fixed for 5 min with 4% paraformaldehyde in Dulbecco’s phosphate buffered saline (DPBS), pH 7.2 (DPBS). Sections were permeabilized with 0.1% Triton X-100 in DPBS for 5 min prior to staining for 4 h at room temperature with 5 μg/mL Alexa 488-labeled wheat-germ agglutinin (Thermo Fisher Inc. [Waltham, MA, USA]) diluted in DPBS. Sections were washed and mounted in Prolong Diamond with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Inc. [Waltham, MA, USA]). Representative images were captured with a Zeiss 710 laser scanning confocal microscope. For image analysis, slides were scanned using a Leica Ariol and automated myofiber cross-sectional area measurements of 1400–1800 myofibers per animal were conducted using Image-Pro Plus image analysis software (Media Cybernetics, Rockville, MD, USA).
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