Under deep pentobarbital anesthesia, mice were transcardially fixed with 4% paraformaldehyde and 10% picric acid in 0.1 M phosphate buffer (pH 7.4). The retinae were dissected and cryoprotected in 30% (wt/vol) sucrose in PBS for >2 h. Pieces of retinae were mounted in OCT compound (Tissue-Tek) and sectioned at a thickness of 10 µm on a cryostat (HM525 NX; Thermo Fisher Scientific). The collected sections were blocked for 1 h in blocking solution (2% normal goat or horse serum, 10% block ace [Dainippon Pharmaceutical], and 0.2% Triton X-100 in PBS) and incubated in primary antibodies in blocking solution overnight at room temperature. The following primary antibodies were used: anti-RIBEYE (1:1,000, rabbit), anti-PKC (1:1,000, mouse; ab31; Abcam), anti-vGluT1 (1:1,000, guinea pig), anti-calbindin (1:2,000, mouse; C9848; Sigma-Aldrich), and anti-Cre (1:800, mouse; MAB3120; and 1:500, rabbit; 69050-3; Millipore). A guinea pig antiserum against vGluT1 was raised against mouse vGluT1 C-terminal 531–560 aa (NM020309; Miyazaki et al., 2003 (link)). The sections were further processed with appropriate Alexa Fluor–conjugated secondary antibodies for 1 h at room temperature. Immunolabeled samples were viewed using a confocal laser microscope (FV1200; Olympus) at room temperature.
Hm525 nx
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The HM525 NX is a high-performance microtome designed for sectioning a wide range of materials. It features a powerful stepper motor-driven drive system and a robust, precision-engineered cutting mechanism to provide consistent, high-quality sections.
Automatically generated - may contain errors
Lab products found in correlation
Tissue tek o c t, by Bio-Optica (4 mentions)
Tissue tek oct compound, by Sakura Finetek (3 mentions)
Fv1200, by Olympus (2 mentions)
Cx41 microscope, by Olympus (2 mentions)
Superfrost plus, by Thermo Fisher Scientific (2 mentions)
Neg 50, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Hematoxylin 7211, by Thermo Fisher Scientific (2 mentions)
Imagej, by Olympus (2 mentions)
Superfrost plus micro slide, by Avantor (2 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (2 mentions)
Nr 616, by BEI Resources (2 mentions)
Nr 614, by BEI Resources (2 mentions)
Anti gfp antibody, by Thermo Fisher Scientific (2 mentions)
Hoechst solution, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 affinipure donkey anti chicken igy igg h l, by Jackson ImmunoResearch (2 mentions)
Dfc7000 gt, by Leica (2 mentions)
Ctr6 led, by Leica (2 mentions)
Mab3120, by Merck Group (1 mentions)
C9848, by Merck Group (1 mentions)
52 protocols using hm525 nx
1
Retinal Immunofluorescence Labeling in Mice
2
Assessing Myocardial Injury and Repair
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After 2 weeks of injection treatment, all the rats were sacrificed, and heart tissues were collected. The hearts were perfused with PBS, embedded in optimum cutting temperature (OCT) compound, and immediately snap frozen in liquid nitrogen. Ten consecutive frozen sections from the left ventricular ligation site to the apex of the left ventricle were prepared at thicknesses of 10 µm with a freezing microtome (HM525 NX, Thermo Fisher Scientific). All sections were stored at −20 °C until they were stained. To assess the size of the injured area, tissue sections were fixed with 4% paraformaldehyde for 2 h and stained with Masson's trichrome. Apoptosis in the myocardial tissue region was analyzed using the DeadEnd™ Fluorescent TUNEL System (G3250, Promega). Immunohistochemistry and immunofluorescence were conducted to examine the expression of active caspase‐3, CD34, Ki67 and Ser1177‐eNOS. All images were captured with a Nikon Ti‐E inverted fluorescence microscope.
3
Immunohistochemical Analysis of Neuronal, Glial, and Microglial Markers
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Brains were sectioned on a cryostat (HM525 NX, Thermo Fisher Scientific, United States) at a thickness of 20 μm. Sections were stained with primary antibodies NeuN, rabbit polyclonal Ab, 1:100 (ABN78, Abcam, United Kingdom), GFAP, mouse polyclonal IgG 1:1000 (63893, Sigma-Aldrich, United States), and Iba1, rabbit polyclonal Ab, 1:500 (019-19741, Wako Chemicals, United States). Secondaries used were goat anti-rabbit IgG 488 (A11036, Invitrogen, United States) for Iba1, and donkey anti-rabbit IgG 647 (A212336, Invitrogen, United States) for NeuN and GFAP, and goat anti-mouse IgG 647 for GFAP (A21236, Invitrogen, United States) all at 1:500 in PBS. Dapi (D1306, Invitrogen, United States) was used for nuclear staining (1:1000) in PBS for 5 min.
4
Histological Analysis of Murine Knee Joints
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Eight weeks after surgery, the mice were sacrificed, and the knee joint samples were fixed with 4% paraformaldehyde (Solarbio, cat. no. P1110) for 24 h. The knee joint samples were then decalcified in EDTA-buffered saline solution (Solarbio, cat. no. E1171, pH = 7.2). The solution was changed every 3 days and the decalcification was completed within 4 weeks. The decalcified samples were embedded in a solution containing equal 30% sucrose and Optimal Cutting Temperature compound (O.C.T. Sakura, cat. no. 4583) for preparing frozen sections. The 10 µm thick sagittal-oriented of the knee joints medial compartment loading area were sectioned in – 20 °C freezing microtomes (HM525NX, Thermo Fisher Scientific) for the following histological analysis.
5
Hypothalamic Protein Localization in Rats
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Successive frozen sections of hypothalamus of 8 μm were made with freezing microtome (HM525NX, Thermo) and the sections were placed in a refrigerator at -80°C. The localization of hypothalamus and ARC in rats refers to the " The Rat Brain in Stereotaxic Coordinates, Compact Third Edition (with CD-ROM)" of Paxinos et al. [27 (link)]. The frozen sections were covered successively with 4% paraformaldehyde for 10 minutes, hydrogen peroxide for 10 minutes, goat serum for 60 minutes, and finally with primary antibodies overnight at 4°C (NPY, catalog no. A3178, ABclonal, China; GHRL, catalog no. A12581, ABclonal, China; GHSR, catalog no. A1840, ABclonal, China); After incubation with secondary antibodies (catalog no. G1215, ABclonal, China) for 1 h at 37°C, the sections were stained with DAB reagent (catalog no. G1212-200, ABclonal, China). All slices were scanned using fluorescence microscope (CKX31, Nikon, Japan).
6
Age-dependent Immunohistochemistry of Rat Muscle
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The EDL and SOL muscles of rats at different age-stages, covered with tissue-tek O.C.T. (Bio-Optica), were frozen in isopentane cooled in liquid nitrogen in a slightly stretched position and stored at −80°C. Serial cross sections (8-μm thick) were cut in a cryostat microtome set at −20°C (HM525 NX, Thermo Scientific). The double-immunofluorescence was peformed as described in previous studies (Nicchia et al., 2016 (link)). The antibodies against ClC-1 and beta-dystroglycan (β-DG) (Novocastra cod: NCL-b-DG) were diluted 1:200 and 1:500, respectively, in PBS-gelation. The antibodies against aquaporin-1 to test the noise background in immunofluorescence was (B-11) sc-25287 (Santa Cruz). Tissue sections were analyzed with a Leica DM RXA epifluorescence microscope and PL Fluotar 16X/0.5na objective (Leica, Heidelberg GmbH, Mannheim, Germany) as previously shown (Nicchia et al., 2016 (link)). For confocal image acquisition and analysis, a Leica TCS SP8 confocal microscope with a 20X and 100X HC PL Apo oil Cs2 objective and Leica LASX software were used (https://www.leica-microsystems.com ).
7
Muscle Fiber Cross-Sectional Analysis
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Gastrocnemius lateralis and soleus muscles, covered with tissue-tek O.C.T. (Bio-Optica), were frozen in isopentane cooled in liquid nitrogen in a slightly stretched position and stored at −80 °C. Serial cross sections (8-μm thick) were cut in a cryostat microtome set a −20 °C (HM525 NX, Thermo Scientific). To measure the cross-sectional area (CSA) of individual fibers, muscle sections were stained for laminin, a major component of the basal lamina, as previously described24 (link). The sections were incubated with primary antibody against rabbit laminin (SIGMA Aldrich c.n. L9393) diluted 1:200 in PBS-gelatin for 1 h. After washing with PBS-gelatin, the sections were incubated with donkey anti rabbit IgG (Invitrogen c.n. A-21206) diluted 1:1000 in PBS-gelatin for 1 h, then washed with PBS-NaCl (300 mM). Sections were examined using Olympus CX41 microscope. Five digital photographs were taken of each muscle section and the CSA was measured as the internal laminin-unstained area by the ImageJ software (NIH, freeware imaging software). For each muscle sample, the number of counted fibers ranged between ∼350 and ∼900 fibers.
8
Cranial Bone Histology Analysis
9
Mass Spectrometry Imaging of Frozen Human Tonsils
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Fresh human tonsils were snap frozen in liquid nitrogen following excision as described above. Tissue sections of 10 μm thickness were prepared using an HM525 NX cryostat (Thermo Scientific, Waltham, MA) operated at −20°C, and thaw-mounted onto the conductive side of indium tin oxide (ITO)-coated slides. Slides were placed in a vacuum desiccator at −10 mmHg for at least 15 min before matrix deposition. Matrix deposition was performed with a TM-Sprayer (HTX Technologies, Chapel Hill, NC). For positive mode mass spectrometry imaging (MSI) experiments, 2,5-dihydroxybenzoic acid (DHB; Alfa Aesar, Ward Hill, MA) was sprayed onto the glass slide after tissue mounting at a concentration of 30 mg mL−1 with 12 passes/cycles (approx. density = 0.001667 mg/mm2). For negative mode MSI, 1,5-diaminonaphthalene (DAN; Sigma-Aldrich, Burlington, MA) was sprayed onto the glass slide after tissue mounting at a concentration of 5 mg mL−1 with 8 passes/cycles (approx. density = 0.009600 mg/mm2). Universal sprayer settings were as follows: nebulization gas pressure = 10 psi, track spacing = 3 mm, velocity = 1200 mm min−1, CC pattern.
10
Quantifying Neuronal Tissue Loss in Mice
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After anesthetized deeply, the mice were perfused intracardially with ice-cold saline and 4% paraformaldehyde. The brain was removed and immersed in 4% paraformaldehyde, 20% sucrose, and 30% sucrose in sequence to complete after-fixing and dehydration. A 25-μm coronal section was collected using the freezing microtome (HM525NX, ThermoFisher).
After washed in PBS and PBS+0.3% Triton, brain sections were incubated in PBS+1% Triton to break the cell membrane and then were blocked with 10% goat serum or donkey serum for 1 h. M.O.M. Kit was used to block mouse antibody. Brain sections were incubated with primary antibodies overnight at 4°C. After washing, the sections were incubated for 2 h at room temperature with secondary antibodies conjugated with Alexa Fluor-488/594/647. DAPI-Fluoromount-G was added and covered the sections with a piece of a clear coverslip.
Ten serial sections with an interval of 11 sections were stained to calculate the volume (mm3) of tissue loss (= the volume of left hemisphere - volume of right hemisphere).
After washed in PBS and PBS+0.3% Triton, brain sections were incubated in PBS+1% Triton to break the cell membrane and then were blocked with 10% goat serum or donkey serum for 1 h. M.O.M. Kit was used to block mouse antibody. Brain sections were incubated with primary antibodies overnight at 4°C. After washing, the sections were incubated for 2 h at room temperature with secondary antibodies conjugated with Alexa Fluor-488/594/647. DAPI-Fluoromount-G was added and covered the sections with a piece of a clear coverslip.
Ten serial sections with an interval of 11 sections were stained to calculate the volume (mm3) of tissue loss (= the volume of left hemisphere - volume of right hemisphere).
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