Culture medium was removed and embedded cultures were washed twice with PBS, prewarmed at 37°C. The matrix was detached from the microwell with a spatula, quickly placed on top of a layer of Tissue Freezing Medium (3801481, Leica Biosystems, Wetzlar, Germany) in a cryomold, and then covered with a second layer of Tissue Freezing Medium to fill the cryomold. The resulting block of cryomatrix containing the cells embedded in the matrix was placed at -80°C until use.
Tissue freezing medium
Manufactured by Leica
Sourced in Germany, United States, United Kingdom, Switzerland, Canada, China
Tissue freezing medium is a laboratory product used to prepare frozen tissue specimens for sectioning and analysis. It is a viscous, clear, or slightly opaque solution that helps maintain the structural integrity of the tissue during the freezing process. The primary function of the tissue freezing medium is to provide a stable, uniform matrix for embedding and supporting the tissue sample during cryosectioning.
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Lab products found in correlation
Cryostat, by Leica (24 mentions)
Cm3050s cryostat, by Leica (10 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (9 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (7 mentions)
Cm3050, by Leica (5 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Vectashield, by Vector Laboratories (4 mentions)
Cm1850, by Leica (4 mentions)
Cm1850 cryostat, by Leica (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Cm1950 cryostat, by Leica (3 mentions)
Lsm 800, by Zeiss (3 mentions)
Hematoxylin and eosin h e, by Merck Group (3 mentions)
Cm1860 cryostat, by Leica (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Cryomicrotome, by Leica (3 mentions)
Superfrost plus, by Thermo Fisher Scientific (3 mentions)
Paraformaldehyde (pfa), by Merck Group (3 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (3 mentions)
Polysine adhesion slide, by Thermo Fisher Scientific (2 mentions)
181 protocols using tissue freezing medium
1
Cryopreservation of Embedded Cultures
2
Histological Analysis of Mouse Muscle and Spinal Cord
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To reduce the numbers of mice in our experiments, considering that a similar phenotype was observed in the Tibialis Anterior (TA) and Gastrocnemius (GA) muscles, TA muscles were consistently used for morphological evaluation of the phenotype. Muscles were dissected, embedded in tissue freezing medium (Leica, Wetzlar, Germany) and frozen in liquid nitrogen pre-cooled isopentane (Sigma-Aldrich, Saint Louis, Missouri, # PHR1661). Cryosections (8 μm or 20 μm) were obtained by using a Leica cryostat.
Ventral spinal cords were isolated from perfused mice, embedded in tissue freezing medium (Leica, Wetzlar, Germany) and frozen in liquid nitrogen pre-cooled isopentane (Sigma-Aldrich, Saint Louis, Missouri, # PHR1661). Cryosections (16 μm) were obtained by using a Leica cryostat.
Hematoxylin and eosin (H&E) (Sigma-Aldrich, Saint Louis, Missouri, # H3136 and # 861006) staining was performed according to the Sigma-Aldrich manufacturer's instructions. Cryosections of spinal cords (16 μm) were also stained with NISSL staining (Sigma-Aldrich, Saint Louis, Missouri, #C5042), performed according to the Sigma-Aldrich manufacturer's instructions.
Ventral spinal cords were isolated from perfused mice, embedded in tissue freezing medium (Leica, Wetzlar, Germany) and frozen in liquid nitrogen pre-cooled isopentane (Sigma-Aldrich, Saint Louis, Missouri, # PHR1661). Cryosections (16 μm) were obtained by using a Leica cryostat.
Hematoxylin and eosin (H&E) (Sigma-Aldrich, Saint Louis, Missouri, # H3136 and # 861006) staining was performed according to the Sigma-Aldrich manufacturer's instructions. Cryosections of spinal cords (16 μm) were also stained with NISSL staining (Sigma-Aldrich, Saint Louis, Missouri, #C5042), performed according to the Sigma-Aldrich manufacturer's instructions.
3
Developing A375 Melanoma Spheroids
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A375, human amelanotic melanoma cells (American Type Culture Collection CRL-1619), were grown in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 25 ng/mL amphotericin B. Cells were cultured at 37°C in a humidified incubator under an atmosphere of 5% CO2 and 95% air. For all experiments, culturing conditions and passage numbers were kept constant.
For the spheroid development, A375 cells that were growing in a monolayer were harvested at second passage and seeded at a density of 1×105 cells per well in a 24-well tissue culture plate precoated with 1.33% agarose.23 (link) Cells were incubated for 72 hours.24 (link) Spheroid development was monitored by microscopy. Spheroids frozen in tissue freezing medium (Leica Biosystems, Concord, Ontario, Canada) were sectioned at 7 µm thickness and stained with eosine for light microscopy to verify stratification.
For the spheroid development, A375 cells that were growing in a monolayer were harvested at second passage and seeded at a density of 1×105 cells per well in a 24-well tissue culture plate precoated with 1.33% agarose.23 (link) Cells were incubated for 72 hours.24 (link) Spheroid development was monitored by microscopy. Spheroids frozen in tissue freezing medium (Leica Biosystems, Concord, Ontario, Canada) were sectioned at 7 µm thickness and stained with eosine for light microscopy to verify stratification.
4
Laser-assisted Microdissection of Banana Embryos
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RNAase free conditions (tools, solutions, handling) were followed throughout the experiment. The experiment was performed as per previously described protocol [52 (link)]. In brief, banana callus (24W) containing embryos were fixed at -23°C under vacuum using tissue freezing medium (Leica biosystems, Germany). Tissue blocks were fixed with the holding clamp of cryomicrotome (Leica biosystems, Germany) and 10–12 μm thin sections were prepared. These sections were taken on slides, air-dried at room temperature and observed under LCM microscope (Zeiss, Germany).
For microdissection, embryos were identified and marked using PALM (Zeiss, Germany) tool. Tissues were snipped-off with a laser beam along with marking in RNase-free tubes and stored at -80°C for RNA isolation.
For microdissection, embryos were identified and marked using PALM (Zeiss, Germany) tool. Tissues were snipped-off with a laser beam along with marking in RNase-free tubes and stored at -80°C for RNA isolation.
5
Immunohistochemical Analysis of Langerin Expression
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Wounds were excised, bisected, and directly frozen in tissue freezing medium (Leica Biosystems, Wetzlar, Germany) without prior fixation. Frozen sections (7 μm) were fixed for 10 min with 4% paraformaldehyde and analyzed by immunohistochemistry staining [39 ] using rat‐anti‐langerin/CD207 IgG2a (eBioL31, Thermo Fisher), the Vectastain ABC peroxidase kit, and the diaminobenzidine peroxidase substrate kit (both from Vector Laboratories, Burlingame, CA).
6
Multiplex RNA in situ Hybridization
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Fresh, unfixed C57BL/6 mouse brains (N = 3) were embedded in tissue freezing medium (Leica Biosystems, Richmond, UK), frozen on a dry ice ethanol bath, sectioned at 20 μm on a cryostat, and then mounted onto Polysine Adhesion Slides (Thermo Fisher Scientific, Waltham, USA). RNA probe hybridization and subsequent washes were performed as described previously56 (link). Fluorescein-labeled probes were detected using peroxidase-conjugated anti-fluorescein antibodies (Roche Diagnostics, Mannheim, Germany). Peroxidase activity was detected using Cy3-tyramide conjugate. After the detection, peroxidase activity was blocked by 100 mM glycine-HCl (pH 2.0) solution containing 0.1% Tween. Sections were washed with 0.1 M Tris-HCl buffer (pH 7.5) containing 0.15 M NaCl and 0.05% Tween and then blocked by 10% goat serum and 1% blocking reagent powder (Roche Diagnostics, Mannheim, Germany) in 0.1 M Tris-HCl (pH 7.5) with 0.15 M NaCl. Dioxygenin (DIG)-labeled probes were detected using peroxidase-conjugated anti-DIG antibodies (Roche Diagnostics, Mannheim, Germany). Peroxidase activity was detected using fluorescein-tyramide conjugate. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, Munich, Germany).
7
Tissue Collection and Processing for α-Syn Analysis
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Tissue was taken from 2-, 3- and 6-month old Line 61 mice and non-transgenic littermates. Mice were deeply anesthetized by Isoflurane (Baxter, Austria) and the thorax was opened to excavate the heart. Animals were flush-perfused transcardially with 0.9% saline through the left ventricle. The hemispheres were divided at midline. One hemisphere was immersion-fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 2 h at room temperature (RT), was then cryoprotected in 15% sucrose in PBS overnight, embedded in tissue freezing medium (Leica Biosystems, Germany) in cryomolds and was snap-frozen in dry ice-cooled liquid isopentane. The frozen samples were then stored at −80 °C until sectioning. The other hemisphere was dissected into hippocampus, striatum and rest brain and shock-frozen on dry ice and stored at −80 °C for α-Syn determination.
8
Tissue Preservation and Sectioning
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Tissue samples were fixed overnight at 4 °C in acetic ethanol (25% acetic acid glacial, 75% ethanol) or 4% paraformaldehyde (PFA) (#P6148, Sigma), embedded in paraffin, and sectioned (3.5 µm thickness). Alternatively, fresh tissue was immediately frozen in tissue freezing medium® (#14020108926, Leica Biosystems, Wetzlar, Germany) and sectioned (5 µm thickness).
9
Zika Virus Infection Dynamics in Neural Progenitor Cells
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NPCs grown on coverslips coated with poly-D-lysine were infected with ZIKV at an MOI of 3 or 0.1, and mock-infected NPCs were used as a control. Coverslips were collected at the indicated time points post-infection and processed for IFA as described previously (Liu et al., 2017 (link)). For cryosections, perfused tissues were fixed in 4% PFA, dehydrated in 30% sucrose, and frozen in tissue freezing medium (Leica Biosystems, Germany). Coronal sections (thickness of 40 μm) were used for immunofluorescence staining as described previously (Tang et al., 2015 (link)). Mouse monoclonal antibodies for DCX (Abcam, Cat#ab18723), Tbr1 (Abcam, Cat#ab31940), Ctip2 (Abcam, Cat#18465) and anti-ZIKV human serum (Ma et al., 2017 (link)) were used as primary antibodies. Secondary antibodies included FITC-anti-mouse-IgG2a (Invitrogen, Cat#11-4210-82), Alexa Fluor 488 goat-anti-human-IgG (Invitrogen, Cat#A-11013) and TRITC-anti-mouse-IgG1 (Southern Biotechnology, Cat#1070-03). Nuclei were counterstained with DAPI (Life Technologies). Images were obtained using a two-photon microscopy with the UltraVIEW VoX 3D Live Cell Imaging System (Perkin Elmer).
10
Salivary Gland Analysis After PET/CT
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After Micro-PET/CT scan, salivary glands were harvested for gene expression analysis and histological study. The qRT-PCR procedures were the same as described above, with the internal control being gapdh. The primers are listed in the key resources table . For histological study, HE staining, AB-Sirius red staining and IF staining of MMP9/CD45 were prepared following the same method as stated above. Samples for succinic dehydrogenase (SDH) and nicotinamide adenine dinucleotide (NADH) staining were instantly immersed in tissue freezing medium (Leica Biosystems, UK) and snap frozen after resection. Cryosections of 5 μm thickness were obtained at −20°C using the CM3050S cryostat (Leica Microsystems, Wetzlar, Germany). For SDH staining, cryosections were incubated with a solution containing sodium succinate, nitro-blue tetrazolium (NBT), and 0.2M phosphate buffer in 37°C water bath for 60 min. Tissue was destained by brief washes in acetone in the following sequence: 30%, 60%, 30% and washed briefly with ddH2O. For NADH staining, the staining solution was prepared by dissolving 1 mg NADH and 1 mg NBT in 1 ml 0.05M Tris-HCl buffer. Sections were incubated with the solution in 37°C water bath for 30 min. Then sections were briefly washed with 60%, 90%, 60% acetone and ddH2O.
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