Late embryonic and early postnatal animals were staged using the EMAP eMouse Atlas Project (http://www.emouseatlas.org) Theiler staging criteria. Temporal bones were collected and fixed in 4% paraformaldehyde (PFA, Polysciences, Inc., Warrington, PA) in 10 mM PBS for 2 hours at room temperature. Cochleae were then dissected using the whole mount method as previously described (Montgomery and Cox, 2016) . For cryosectioning, PFA-fixed inner ears were put through a sucrose gradient (10% sucrose for 30 minutes, 15% sucrose for 30 minutes, and 30% sucrose overnight), submerged in OCT (Tissue-Tek, Sakura Finetek USA, Torrance, CA) and flash frozen. Sections cut at 14 µm thickness were collected on SuperFrost Plus slides (Fisher, Hampton, NH).
Paraformaldehyde (pfa)
Manufactured by Sakura Finetek
Sourced in United States, Japan
Paraformaldehyde is a white, crystalline solid that is used as a fixative and preservative in various laboratory applications. It is a polymer of formaldehyde and is commonly used to preserve and stabilize biological samples for microscopy and other analytical techniques. Paraformaldehyde is highly reactive and is often used to cross-link proteins and nucleic acids, which helps to maintain the structural integrity of samples.
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Lab products found in correlation
Paraformaldehyde (pfa), by Merck Group (3 mentions)
Oct compound, by Sakura Finetek (2 mentions)
Optimal cutting temperature compound, by Sakura Finetek (2 mentions)
Optimum cutting temperature compound, by Sakura Finetek (2 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (1 mentions)
Tissue tek, by Sakura Finetek (1 mentions)
Paraformaldehyde (pfa), by Polysciences (1 mentions)
Isolectin gs ib4, by Thermo Fisher Scientific (1 mentions)
Lsm 800 confocal scanning microscope, by Zeiss (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Tissue tek optimal cutting temperature compound, by Sakura Finetek (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
Paraformaldehyde (pfa), by Sinopharm (1 mentions)
Proliferating cell nuclear antigen (pcna), by Abcam (1 mentions)
Alexa fluor 488 goat anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
Cd31 mouse monoclonal antibody, by Abcam (1 mentions)
Nestin, by Abcam (1 mentions)
Goat anti mouse igg h l cy3, by Jackson ImmunoResearch (1 mentions)
9 protocols using paraformaldehyde (pfa)
1
Staging and Sectioning of Mouse Temporal Bones
2
Retinal Dissection and Immunostaining
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Retinal dissection was carried out as previously described, 37 and whole-mounted retinas were preserved in 0.4% paraformaldehyde (PFA) (Sigma). Enucleated eyes were fixed with 4% PFA and embedded in Tissue-Tek optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA). Before immunostaining, whole-mounted retinas and cryosections (12 μm, Leica CM1950) were rinsed in PBS (Sigma, St. Louis, MO, USA) three times (5 min/time) and blocked in PBS containing 5% FBS (Invitrogen, Waltham, MA, USA) and 0.2% Triton X-100 for 30 minutes at room temperature, followed by incubation with primary antibodies at 4 °C overnight. The following primary antibodies were diluted in blocking buffer: Isolectin GS-IB 4 (1:100; Invitrogen, Waltham, MA; I21411). Then, the sections were washed three times with PBS and labeled for 1-4 hours with Alexa Fluor™-488-or Alexa Fluor™-594-labeled goat antirat or antirabbit IgG or donkey antigoat IgG secondary antibody (1:500; Invitrogen). Images were captured on a LSM 800 confocal scanning microscope (Zeiss, Thornwood, NY, USA). The person who was assigned to cell counting was blind to the genotypes of the samples. No animals were excluded from this study.
3
Brain Tissue Preparation for Molecular Analysis
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At different time points after LPS/saline injection, mice were rapidly euthanized by cervical dislocation, minimizing suffering, discomfort, or stress. To measure curcumin concentration, immediately after sacrifice, blood samples were collected by cardiac puncture for separation of plasma, which was stored at -80°C. Animals were then decapitated, whole brains removed and immediately used or frozen in liquid nitrogen and stored at -80°C until analysis.
To measure mRNA expression of target genes after decapitation, brain regions (cortex, striatum, hippocampus, and cerebellum) were dissected out based on stereological coordinates using a mouse brain atlas (Franklin and Paxinos, 2007 ; Sorrenti et al., 2018 (link)), frozen in liquid nitrogen and stored at -80°C until further analysis.
For immunofluorescence studies, mice were anesthetized with isoflurane and perfused via the cardiovascular system with saline followed by 4% paraformaldehyde (Sigma-Aldrich). Brains were removed intact, post-fixed with 4% paraformaldehyde for 2 h at 4°C, dehydrated through an ascending sucrose series (15 and 30% sucrose) at 4°C overnight and then embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA, United States). The embedded brains were kept at -80°C until sectioning.
To measure mRNA expression of target genes after decapitation, brain regions (cortex, striatum, hippocampus, and cerebellum) were dissected out based on stereological coordinates using a mouse brain atlas (Franklin and Paxinos, 2007 ; Sorrenti et al., 2018 (link)), frozen in liquid nitrogen and stored at -80°C until further analysis.
For immunofluorescence studies, mice were anesthetized with isoflurane and perfused via the cardiovascular system with saline followed by 4% paraformaldehyde (Sigma-Aldrich). Brains were removed intact, post-fixed with 4% paraformaldehyde for 2 h at 4°C, dehydrated through an ascending sucrose series (15 and 30% sucrose) at 4°C overnight and then embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA, United States). The embedded brains were kept at -80°C until sectioning.
4
Kidney Time-Course Histology Protocol
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Rats were anesthetized and sacrificed, and kidneys were removed at the indicated time points (0, 1, 2, 4, 7, 14, 28 days post-resection). For paraffin sections, kidneys were fixed with 4% (vol/vol) paraformaldehyde (Wako Pure Chemical Industries, Ltd., Osaka, Japan), paraffin-embedded, and cut into 4 μm sections. For frozen sections, kidneys were fixed with 4% paraformaldehyde for 2 h on ice, incubated in 30% (vol/vol) sucrose in PBS at 4 °C for overnight, embedded in optimum cutting temperature compound (Sakura FineTek Japan co., Ltd., Tokyo, Japan), and cut into 5 μm sections.
5
Lung Histomorphometry in Mice
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The mice (n=8), used for morphometric analyses were anesthetized with 10% chloral hydrate (0.03 ml/10 g) and sacrificed by exsanguination of the abdominal aorta. The lungs were isolated and fixed by infusion with 4% paraformaldehyde (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) through a tracheal cannula at a constant pressure of 25 cm H2O, part of the left lung was frozen and embedded in OCT Compound (#4583; Sakura Finetek USA, Inc., Torrance, CA, USA), and other parts were then immersed in paraformaldehyde. Between 24 and 48 h later, a mid-sagittal slice of the left lung was embedded in paraffin (Kangtai Clinical Reagent Co., Ltd., Beijing, China), and 5 μm sections were dewaxed and hydrated prior to histological analyses. The sections were then stained with hematoxylin and eosin and Sirius Red (Sigma-Aldrich China, Inc., Shanghai, China) for detection of collagen fibers.
6
Subcutaneous Membrane Implantation in Mice
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The animal experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Asan Medical Center (protocol number: 2016-02-234). All institutional and national guidelines for the care and use of laboratory animals were followed. Eight-week-old male nude mice were housed in an animal room in a specific pathogen-free facility with controlled temperature and relative humidity.
For implantation, mice were anesthetized with isoflurane, and two subcutaneous pockets were made to both the left and the right of the midline of the backs of the mice via 1.5 cm incisions (Figure 1 B, n = 16 mice). The multilayered membranes were implanted in the subcutaneous pockets. The implanted tissue sheets were harvested with the surrounding tissue 1, 2, 4, or 8 weeks after implantation and then fixed in 4% paraformaldehyde at 4 °C overnight and embedded in optimal cutting temperature (OCT) compound (Sakura Finetek USA INC., Torrance, CA, USA), frozen, and stored at –80 °C. We subsequently used a cryostat to obtain 10 µm sections, which were stained using standard hematoxylin and eosin (H&E) staining methods. Sections were viewed using routine bright-field light microscopy.
For implantation, mice were anesthetized with isoflurane, and two subcutaneous pockets were made to both the left and the right of the midline of the backs of the mice via 1.5 cm incisions (
7
Quantifying Angiogenic Effect of M2 Exosomes on SCI
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To assess the angiogenic effect of the M2 macrophage exosomes on the SC of the rats post-SCI, the proportion of proliferating blood vessels was detected by immunofluorescence staining. After SCI for 3 days, the SC tissues were collected and fixed with paraformaldehyde (4%) for 3 h. After incubation with 6% sucrose in PBS overnight, an optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA) was used to embed the tissues and then sectioned at 5 μm thickness (n = 5/group). After air-drying for 15 min, 10% normal goat serum/PBS was added to the sections for 1 h, and then, they were exposed to proliferating cell nuclear antigen (PCNA, Shanghai, China) rabbit monoclonal antibody (1:200; Abcam, Cambridge, UK) or CD31 mouse monoclonal antibody (1:100; Abcam, Cambridge, UK), Nestin (1:200, Abcam, Cambridge, UK), NeuN (1:200, Abcam, Cambridge, UK), and Sox2 (1:200, Sangon Biotech, Shanghai, China) overnight at 4 °C. After washing with PBS, the sections were exposed to the secondary antibody Cy3 goat anti-mouse IgG (H + L) (1:1000, Jackson, Bar Harbor, ME, USA) or Alexa Fluor 488 goat anti-rabbit IgG (H + L) (1:1000, Jackson, Bar Harbor, ME, USA) for 30 min. The sections were rinsed thrice with PBS and exposed to DAPI solution (CST, Boston, MA, USA) for 15 min. The number of positive cells in the SC was counted.
8
Histopathological and Immunofluorescence Analysis of Lung Tissues
9
Immunofluorescence Analysis of EpH4 Cell Aggregates
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EpH4 cells were similarly cultured with MM, GM, or matrigel-coated GM to form the cell aggregates. After incubation for 7 days, the cell aggregates were fixed with 4 vol% paraformaldehyde at 4 °C for 1 h and embedded in optimal cutting temperature compound (Sakura Finetek Japan Ltd, Tokyo, Japan) and frozen in liquid nitrogen. The frozen samples were sectioned using a cryotome (CM3050S, Leica Microsystems, Wetzlar, Germany) and incubated at 4 °C overnight with the following primary antibodies: β-casein (Santacruz Inc., America, 1:50) or Laminin (Abcam Inc., Cambridge, UK, 1:100). Then, secondary antibodies coupled to Alexa 488 (Molecular Probes, Invitrogen Inc., Carlsbad, America, 1:700) were incubated at 25 °C for 30 min for fluorescent viewing. Next, TO-PRO-3 (Molecular Probes, Eu-gene, USA) was added to incubate at 25 °C for 10 min for the nuclear staining of cells. The sections of 10 μm thickness were viewed in a confocal laser scanning microscope (FV1000D, Olympus Ltd, Tokyo, Japan).
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