The tumors were immersed in Optimum Cutting Temperature™ compound (Leica Biosystems, Richmond, CA, USA), frozen in liquid nitrogen, and sectioned at a 5 µm thickness. The sections were permeabilized, blocked for 60 min with 10% goat antiserum and 0.25% Triton X-100 in phosphate-buffered saline to prevent nonspecific binding, and subsequently incubated with an anti-CD31 (1:200), anti-VEGFR2 (1:200), anti-E-cadherin (1:100), anti-N-cadherin (1:100), anti-snail (1:100), or anti-vimentin (1:100) antibody. Next, the sections were washed and incubated with the corresponding Alexa-488-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Fluorescence images were acquired using a confocal microscope (Carl Zeiss, Oberkochen, Germany). The fluorescent intensity was acquired by image analysis of Zen software provided by Carl Zeiss.
Opti mum cutting temperature compound
Manufactured by Leica
Sourced in Germany, China
Opti-mum Cutting Temperature compound is a specialized media used for preparing tissue samples for microscopic analysis. It is designed to maintain the optimal cutting temperature during the sectioning process, ensuring high-quality tissue sections for histological examination.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Pen membrane covered slides, by Zeiss (1 mentions)
Lsm 880, by Zeiss (1 mentions)
Cy5 conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Zen software, by Zeiss (1 mentions)
Anti cd31, by BD (1 mentions)
Anti α sma, by Abcam (1 mentions)
Cy5 or cy2 conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Cm1950, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Rat antimouse cd31 primary antibodies, by BD (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
7 protocols using opti mum cutting temperature compound
1
Immunofluorescence Imaging of Tumor Markers
2
Cryogenic Preservation of Mouse Mandibular Condyles
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Animal use protocol was approved by Columbia University Institutional Animal Care and Use Committee (IACUC). Mandibular condyles of postnatal 7-day CD-1 mice (Charles River Laboratory, Stone Ridge, NY) were surgically removed after inhalational anesthesia and immediately embedded in optimum cutting temperature compound (Leica Biosystems), frozen on dry ice, and stored at −80°C until sectioning. Frozen sections (10-μm thickness) were prepared using a cryostat at −20°C and were mounted on precooled PEN membrane-covered slides (Zeiss). Slides were stored at −80°C until further processing.
3
Tumor Vessel Characterization Protocol
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The tumors were collected and fixed in 4% paraformaldehyde at 4 °C for 8 h and dehydrated by soaking the tissues blocks in 20% and 30% sucrose solution for 24 h respectively. The tissues were embedded in optimum cutting temperature compound (Leica, Weztlar, Germany). To determine the vessel braches and calculate the tumor microvascular density, the sections of 100 μm were incubated with anti-CD31 (1:100, BD Biosciences, Franklin Lakes, NJ, USA) at 4 °C overnight and then incubated with Cy5-conjugated secondary antibody (1:200, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at 37 °C. Z-stacks of tumor vessels were acquired with confocal microscope (LSM 880, Zeiss) and three-dimensional reconstruction of the tumor vessels was accomplished with ZEN software (Zeiss). For the pericyte coverage experiment, the sections of 12 μm were incubated with anti-CD31 (1:100, BD Biosciences) and anti-α-SMA (1:100, Abcam, Cambridge, UK) overnight and then incubated with Cy5 or Cy2-conjugated secondary antibody (1:200, Jackson ImmunoResearch) for 1 h at 37 °C. The pericytes around tumor vessels were observed with confocal microscope (LSM 880, Zeiss) using airscan. For the quantitative analyses, at least two random optical fields per tumor section were captured.
4
DOPA Staining of Tissue Sections
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The tissues were embedded in OCT (Opti-mum Cutting Temperature compound, Leica, Germany) to prepare 7-μm serial sections. The sections were stained according to the method described by Rui [20 ], with the following modifications: frozen sections were equilibrated to room temperature, rinsed in distilled water, and incubated in DOPA buffer containing 1.104 g of DOPA (Sigma-Aldrich, Germany) in 1 L of 0.01 mol/L phosphate-buffered saline (PBS) for 30 min at 37°C. The sections were then incubated in fresh DOPA buffer for 30 min at 37°C or until the color of the buffer darkened. After counterstaining with 1% Nuclear Fast Red buffer for 10 min at room temperature, the sections were dehydrated in 100% alcohol and xylene, and then they were mounted for observation using a light microscope. Stained cells exhibited black cytoplasm. Control staining reactions were performed using PBS instead of DOPA.
5
Histological Analysis of Caecum Lipid Content
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After fixation in 4% paraformaldehyde solution for a minimum period of 24 h before use, the caeca were trimmed and dehydrated by 30 and 50% sucrose solution, and then embedded in OCT (Opti-mum Cutting Temperature compound, Leica, Shanghai, China) to prepare 15 mm frozen sections. Sections were washed with distilled water and incubated in oil red O for 10 min. After being rinsed with isopropanol for 2 s and distilled water for 1 s, the sections were rinsed with Hematoxylin solution for 5 min, and then incubated in distilled water for 10 min. And then the sections were mounted with neutral balsam for observation under light microscope. The histopathological changes were observed and pictured using a Zeiss camera system (Carl Zeiss Optics Co., Ltd., Guangzhou, China).
6
Tumor Vasculature Immunostaining Protocol
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The tumor nodules were dissected and frozen in optimum cutting temperature compound (Leica, Wetzlar, Germany) immediately after treatment. The tumors were cryosectioned (10 μm; Leica CM1950), and all slides were stored at −80°C before staining. The frozen optimum cutting temperature sections were fixed in acetone for 10 minutes and stained with rat antimouse CD31 primary antibodies (BD Biosciences, Franklin Lakes, NJ, USA). Donkey antirat Alexa Fluor® 488 (Invitrogen, Waltham, MA, USA) was used as a secondary antibody. A negative control was performed by omitting the primary antibody and incubating with secondary antibody only. The sections were washed twice and mounted with medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA).
7
Immunofluorescence Bax Protein Detection
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The fixed BFs were trimmed and dehydrated by 30% and 50% sucrose solution, and then embedded in OCT (Opti-mum Cutting Temperature compound, Leica, Shanghai, China) to prepare 5 µm frozen sections. Sections were washed with distilled water and then incubated in 3% hydrogen peroxide solution (Beijing Solarbio Life Science and Technology Co., LTD, Beijing, China) for 15 min at room temperature. After washed with the distilled water, the sections were blocked with 1% bovine serum albumin (Beijing Solarbio Life Science and Technology Co., LTD, Beijing, China) for 20 min at room temperature. The sections were incubated with primary monoclonal antibody rabbit anti-Bax (Santa Cruz, TX, 1:200) overnight at 4°C. After rinsed with PBS, sections were reacted with goat antirabbit IgG conjugated with fluorescein isothiocyanate (Zymed Laboratories Inc, Beijing, China) at room temperature for 1 h. After rinsing with PBS, the sections were observed and pictured using Zeiss camera system (Carl Zeiss Optics Co. LTD, Guangzhou, China). In the negative control, the primary antibody was replaced with PBS.
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