Tissue fixation and staining of sections or tissues with antibodies was performed by the following method. Freshly-excised femoral or tibial bone from WT mice was embedded in super cryoembedding medium (Leica Microsystems) and frozen in liquid nitrogen. Next, 10-μm cryosections were generated via the Kawamoto film method54 (link). An anti-c-Kit (AF1356, R&D Systems, dilution 1/100), anti-Sca1 (553333, BD Biosciences, dilution 1/100), anti-CD71 (113802, Biolegend, dilution 1/100) and, anti-Regnase-1 antibodies (dilution 1/100)16 (link) were used for staining55 (link). Cell nuclei were visualized with TO-PRO-3 and sections were examined by confocal microscopy (TCS/SP8; Leica). EdU labeling and staining was performed using a Click-iT® Plus EdU Alexa Fluor 555 Imaging Kit (Thermo Fisher) according to the manufacturer’s instructions. In all assays, isotype-matched Ig was used as a negative control and it was confirmed that the positive signals were not derived from nonspecific background. Images were processed using Photoshop CS2 software (Adobe Systems). All images shown are representative of 3 to 5 independent experiments.
Super cryoembedding medium
Manufactured by Leica
Sourced in Germany
Super Cryoembedding Medium is a specially formulated medium used for the preparation of cryosections. It is designed to provide optimal support and protection for delicate tissue samples during the freezing and sectioning process.
Automatically generated - may contain errors
Lab products found in correlation
Axiovert 200m, by Zeiss (2 mentions)
Af1356, by R&D Systems (1 mentions)
Click it plus edu alexa fluor 555 imaging kit, by Thermo Fisher Scientific (1 mentions)
Tcs sp8, by Leica (1 mentions)
Trap alp staining kit, by Fujifilm (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Technovit 9100, by Kulzer (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Fluorescence microscope, by Keyence (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Fluoview software, by Olympus (1 mentions)
Dmire2 confocal laser scanning microscope, by Leica (1 mentions)
Prolong glass antifade mountant, by Thermo Fisher Scientific (1 mentions)
Donkey anti goat igg h l, by Jackson ImmunoResearch (1 mentions)
Rm2235, by Leica (1 mentions)
Nex c3, by Sony (1 mentions)
5 protocols using super cryoembedding medium
1
Cryosectioning and Immunostaining of Murine Bone
2
Maxillary Bone Analysis Protocol
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Rat maxillary bones were dissected and frozen in hexane cooled with carbon dioxide, followed by embedding in the media (Supercryo-embedding medium, SCEM; Leica Microsystems, Wetzlar, Germany). SCEM frozen blocks were covered with adhesive film (Cryofilm Type 2C; Leica) and coronally sectioned with the film at 5 μm intervals. Sections were then fixed in 10% neutral buffer formalin solution and stained with Mayer's hematoxylin and eosin. ALP activity was evaluated using a TRAP-ALP staining kit (Wako). To visualize bone double labeling, rat maxillary bones were dissected, fixed with 70% ethanol, and dehydrated in an ethanol series. Samples were then embedded with methyl methacrylate resin (Technovit9100; Heraeus-Kulzer, Hanau, Germany) and coronally sectioned every 40 μm (Kureha Special Laboratory, Tokyo, Japan). Sections were stained with toluidine blue and observed under an inverted optical microscope (Axiovert 200M; Carl Zeiss, Jena, Germany) using Axio Vision software (version 4.8; Carl Zeiss). The number of nuclei was counted on HE-stained images, at the suture, the osteogenic front, and the newly formed bone area (1000 × 1000-pixel box over the MPS).
3
Immunohistochemical Analysis of IL-17A and γδ TCR
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For immunohistochemical analysis, hindlimbs were embedded in Super Cryoembedding Medium (Leica Microsystems Japan, Tokyo, Japan), and frozen sections (5 μm) were generated. The sections were fixed in cold acetone for 5 min and blocked with 4% bovine serum albumin (Sigma, St Louis, MO, USA) and 5% goat serum in PBS. Antibodies used were as follows: 2 μg ml−1 rabbit anti-mouse IL-17A polyclonal Ab (Abcam, ab9565-100, Cambridge, UK), 5 μg ml−1 hamster anti-mouse γδ TCR mAb (GL3, BD Pharmingen, San Diego, CA, USA), 2.5 μg ml−1 Alexa Fluor 488-goat anti-rabbit-IgG (Life Technologies, Carlsbad, CA, USA), and 2.5 μg ml−1 Cy3-goat anti-hamster IgG (Jackson ImmunoResearch, West Grove, PA, USA). Nuclei were stained with 0.5 μg ml−1 4,6-diamidino-2-phenylindole. The slides were visualized on a fluorescence microscope (Keyence, Osaka, Japan) and on an Olympus FV1000 Confocal Microscope, operated by the FluoView software (Olympus, Tokyo, Japan).
4
Immunofluorescence Staining of Respiratory Syncytial Virus
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Lung and nasal cavity samples for confocal microscopy were prepared as described previously91 (link). Briefly, the samples were fixed in 4% (wt/vol) paraformaldehyde in PBS overnight at 4 °C with rocking, followed by soaking in 20% (wt/vol) sucrose in PBS overnight at 4 °C with rocking. The samples were then embedded in Super Cryoembedding Medium (Leica Microsystems K.K., Wetzlar, Germany). For immunofluorescence staining, 10-μm-thick frozen sections were prepared by using a CryoJane Tape-Transfer System (Instrumedics, St. Louis, MO, USA) and allowing the sections to air dry. The RSV in the sections was detected by using a goat anti-RSV polyclonal antibody (1:480; #AB1128, Millipore, Billerica, MA, USA) and a donkey anti-goat IgG (H+L) (1:400; #705-166-147, Jackson ImmunoResearch, West Grove, PA, USA). Nasal cavity sections were further stained for actin filament with Phalloidin-iFluro647 Reagent (1:1000; #176759, Abcam). After washing, the specimens were mounted in ProLong Glass Antifade Mountant (Thermo Fisher Scientific) with DAPI (Vector Laboratories, Burlingame, CA, USA) and analyzed by using a DM-IRE2 confocal laser-scanning microscope (Leica Microsystems K.K.).
5
Wound Evaluation: Macroscopic and Microscopic
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Macroscopic evaluation: Wound photos with scale indication were obtained from directly above with a digital camera (NEX-C3, Sony, Tokyo, Japan). The wound area was measured using a computer-assisted morphometric analyzer (VH Analyzer, VH-H1A5, KEYENCE Co., Osaka, Japan).
Microscopic evaluation: Dorsal skin tissue was dissected from the sacrificed mice. This tissue, together with the splint, was fixed in 10% buffered formaldehyde (pH 7.4) for several days. Two cross-cut tissue samples from each wound (about 5 mm thick) were excised (Sample-1 and Sample-2). Paraffin blocks were prepared by using a tissue processor (Tissue-Tec VIP Premier, SAKURA, Nagano, Japan), following which, 4-μm-thick tissue sections were cut with a microtome (RM2235, Leica Biosystems, Nußloch, Germany). For the frozen section, the Kawamoto method was used [22 (link)]. After fixing with 5% neutral formalin, tissue was embedded in super cryo-embedding medium (Leica Microsystem GmbH, Wetzlar, Germany) and then quickly frozen in liquid nitrogen. After adhesion of cryofilm (Leica Microsystem GmbH, Wetzlar, Germany) on the frozen block, a 4 µm-thick section was cut with a cryostat (CM350: Leica Microsystem GmbH, Wetzlar, Germany).
Microscopic evaluation: Dorsal skin tissue was dissected from the sacrificed mice. This tissue, together with the splint, was fixed in 10% buffered formaldehyde (pH 7.4) for several days. Two cross-cut tissue samples from each wound (about 5 mm thick) were excised (Sample-1 and Sample-2). Paraffin blocks were prepared by using a tissue processor (Tissue-Tec VIP Premier, SAKURA, Nagano, Japan), following which, 4-μm-thick tissue sections were cut with a microtome (RM2235, Leica Biosystems, Nußloch, Germany). For the frozen section, the Kawamoto method was used [22 (link)]. After fixing with 5% neutral formalin, tissue was embedded in super cryo-embedding medium (Leica Microsystem GmbH, Wetzlar, Germany) and then quickly frozen in liquid nitrogen. After adhesion of cryofilm (Leica Microsystem GmbH, Wetzlar, Germany) on the frozen block, a 4 µm-thick section was cut with a cryostat (CM350: Leica Microsystem GmbH, Wetzlar, Germany).
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