At endpoint, lungs were harvested, taking care to preserve the trachea, and fixed in 2% PFA for 2 hours on ice. Tissues were cryopreserved in 30% sucrose overnight at 4°C. Lungs were inflated through the trachea with a mixture of 50% OCT Compound (Fisher Scientific) in PBS and flash frozen in OCT Compound in cryomolds. For each mouse, a minimum of three 6μm sections taken at least 100μm apart were stained with hematoxylin and eosin (H&E) for histologic analysis. Sections of 20μm were used for immunofluorescence staining. See Supplementary Materials and Methods .
Oct compound
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom
The OCT compound is a high-performance optical coherence tomography (OCT) reagent designed for use in biomedical and scientific research applications. It is a versatile compound that can be used to enhance the imaging and analysis capabilities of OCT systems. The core function of the OCT compound is to provide improved signal-to-noise ratio and contrast in OCT imaging, enabling researchers to obtain more detailed and accurate information about the samples being studied.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (13 mentions)
Cryostat, by Leica (11 mentions)
Paraformaldehyde (pfa), by Merck Group (6 mentions)
Triton x 100, by Merck Group (6 mentions)
Superfrost plus microscope slide, by Thermo Fisher Scientific (5 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (5 mentions)
Cm3050, by Leica (5 mentions)
Cm3050s cryostat, by Leica (5 mentions)
Cm1850 cryostat, by Leica (5 mentions)
Cm1950 cryostat, by Leica (5 mentions)
Fv3000 confocal microscope, by Olympus (4 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (4 mentions)
Fluoromount g, by Southern Biotech (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (4 mentions)
Lsm 880 confocal laser scanning microscope, by Zeiss (3 mentions)
Prolong gold, by Thermo Fisher Scientific (3 mentions)
Sucrose, by Merck Group (3 mentions)
Superfrost slide, by Thermo Fisher Scientific (3 mentions)
Superfrost plus slides, by Avantor (3 mentions)
251 protocols using oct compound
1
Lung Tissue Cryopreservation and Sectioning
2
Glioma Xenograft Models for Therapeutic Testing
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All experiments using animals were performed under the Institutional Animal Care and Use Committee-approved protocol at Northwestern University in accordance with NIH and institution guidelines. Athymic (Ncr nu/nu) female mice at an age of 6–8 weeks (Taconic Biosciences) were used for all animal experiments. Patient-derived glioma spheres (5 × 104 or 5 × 105 cells in 2 or 5 μl PBS) were stereotactically implanted into the brains of individual mice, with five mice per group. The glioma-bearing mice were euthanized 2–6 weeks after implantation. The brains were removed, processed and analysed as we previously described55 (link). For subcutaneous tumour xenograft model, 1 × 106 cells of PN or MES glioma spheres in 100 or 50 μl PBS were injected into the flanks of nude mice. When palpable tumour xenografts formed, the tumour-bearing mice at every third day were treated by intraperitoneal with LGK974 (3 mg kg−1)37 (link), BAY-117082 (10 mg kg−1, 5 μM)38 (link) or vehicle (0.5% MC/0.5% Tween 80). Mice were euthanized when tumour sizes reached ∼1,500 mm3 or pathological symptoms were developed. The flank xenografted tumours were then removed, removed, embedded in OCT compound (Thermal Fisher), and stored at −80 °C. Tissues of xenografted tumours were sectioned on a cryostat in 8-μm thickness and stained with haematoxylin and eosin or IHC as described above55 (link).
3
Brain Tissue Preparation for IHC
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After the behavioral tests, the animals were euthanized, and cervical dislocation was performed. The brains were removed, separated into two hemispheres, and immersed in 30% sucrose for 3 h at 4 °C. They were then embedded in optimal cutting temperature (OCT) compound (Themo Fisher Scientific, Karlsruhe, Germany), frozen immediately in liquid nitrogen cooled isopentane, and stored at −80 °C for immunohistochemistry.
4
Brain Tissue Cryopreservation Protocol
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The day after NOR test, rats were sacrificed. Their brains were cut to obtain two hemispheres and immersed in a cryoprotectant containing 30% sucrose solution at 4 °C for 3 h. Then, optimal cutting temperature (OCT) compound (Themo Fisher Scientific, Darmstadt, Germany) was used to fix each hemisphere. After that, each hemisphere was promptly immersed in liquid nitrogen-cooled isopentane (Sigma-Aldrich, Inc., St. Louis, USA). Finally, the hemispheres were kept at −80 °C for immunohistochemical study.
5
Immunostaining of Human Cerebral Organoids
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Human cerebral organoids were fixed in 4% paraformaldehyde in Phosphate-Buffered Saline (PBS) overnight at 4°C, dehydrated with 30% sucrose in PBS and embedded in O.C.T. Compound (Thermo Fisher Scientific, Waltham, MA, USA). Cryostat sections (14 μm) were cut and mounted on slides (Thermo Fisher Scientific, Waltham, MA, USA). Mounted sections were incubated at room temperature for 1 h with blocking solution [3% normal goat serum + 0.3% Triton X-100 in tris-buffered saline (TBS)] and subsequently incubated with primary antibodies diluted in blocking solution overnight at 4°C. Antibodies specific for TUJ1 (1:400, T8660, Sigma-Aldrich, St. Louis, Missouri, USA) were used for immunostaining. After three washes with TBS, corresponding fluorescent dye Alexa Fluor 488-conjugated anti-mouse IgG (715-545-151, Jackson Immunoresearch, West Grove, PA, USA) secondary antibodies diluted in the blocking solution were added and samples were incubated at room temperature for 2 h and followed by 4′,6-diamidino-2-phenylindole (DAPI) (Vectashield Mounting Medium with DAPI, H-200; Vector Laboratories, Burlingame, CA, USA) staining. Finally, stained slides were rinsed with TBS three times, mounted, and analyzed using a FV3000 Confocal Microscope (Olympus, Shinjuku, Tokyo, Japan).
6
Immunolocalization of Schistosoma Antigens
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Live, 7-day-old schistosomula were fixed in 4% paraformaldehyde (PFA) for 5 min at room temperature. The fixed parasites were incubated first with blocking buffer (1% bovine serum albumin (BSA) in PBS) for 1 h and then in serum (diluted 1:50 in blocking buffer) from either rSmPGM-immunized mice or control mice, for 1 h at room temperature. The worms were then washed with PBST before being incubated for 1 h in the dark at room temperature with Alexa FluorTM 488 goat anti-mouse IgG (H + L) (Invitrogen) diluted in blocking buffer at 1:100. The worms were again washed with PBST and then incubated with 0.3 mM 4’,6-diamidino-2-phenylindole (DAPI) in blocking buffer for 5 min, washed with PBST, mounted in Fluoromount, and placed in clear-bottom 96-well plates. The parasites were viewed using an inverted fluorescent microscope (Eclipse Ti, Nikon and Revolve, Echo).
Frozen sections (6 μm thick) of adult parasites in optimal cutting temperature (OCT) compound (Thermo Fisher Scientific) were hydrated with PBS and incubated with blocking buffer for 1 h at room temperature in a humidified chamber. The sections were processed for immunolocalization using the same techniques described above for whole, fixed schistosomula.
Frozen sections (6 μm thick) of adult parasites in optimal cutting temperature (OCT) compound (Thermo Fisher Scientific) were hydrated with PBS and incubated with blocking buffer for 1 h at room temperature in a humidified chamber. The sections were processed for immunolocalization using the same techniques described above for whole, fixed schistosomula.
7
Bone Structural Analysis of CD97 Knockout Mice
8
Immunofluorescent Labeling of Mouse Brain
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Brains were harvested from postnatal day 21 mice and fixed for 48 h in 4% paraformaldehyde followed by 48 h in 30% sucrose in 1× PBS. Brain was then cut down the midline before freezing in OCT compound (optimum cutting temperature compound; catalog #23-730-571, Thermo Fisher Scientific). A cryostat was used for slide mounting 10 μm sagittal sections of brain tissue. Slides were stored at −80°C.
Slides were incubated in a blocking solution (PBS, 5% donkey serum, 0.1% Triton-X 100) for 1 h in a humidified chamber at room temperature, then with chicken anti-GFP primary antibody (1:1000; RRID:AB_10000240 ) in blocking solution overnight in humidified chamber at 4°C. Following washes in PBS, slides were incubated in donkey anti-chicken Alexa Fluor-488 secondary antibody (diluted 1:400 in blocking solution). Slides were washed with PBS, incubated with DAPI, washed again, and mounted with Prolong Gold.
Slides were incubated in a blocking solution (PBS, 5% donkey serum, 0.1% Triton-X 100) for 1 h in a humidified chamber at room temperature, then with chicken anti-GFP primary antibody (1:1000; RRID:
9
Comprehensive Tissue Sampling and Preservation
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Under deep isoflurane anesthesia, mice were decapitated before dissection of brain, cochlea, and internal organs. Whole brains were directly transferred into 4% PFA and left 24 h at +4°C for fixation. After fixation, brains were immersed in 10%, 20%, and 30% cryoprotective sucrose solutions for 24 h at +4°C, respectively. After that, brains were completely embedded in cube molds with OCT compound (Thermo Fisher Scientific) and flash-frozen in liquid nitrogen for storage at −80°C. After brain removal, inner and middle ear structures, that is, cochlea, vestibular system and the ossicular chain, were gently prepared from the respective temporal bones using fine-tip forceps. The protective bony bulla was also removed to visualize the snail shape and round window of the cochleae for sampling. Moreover, tissue samples of a representative organ spectrum including the heart, kidney, liver, lung, and spleen were collected. Each sample was divided with one fraction being frozen at −20°C for PCR analysis and the other fixed in 4% neutral buffered formalin (FA) for histology.
10
Capillary Quantification in Cardiac Tissue
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At the time of sacrifice, hearts were perfused with GFP-conjugated Isolectin B4 from Griffonia simplicifolia (Vector Lab) for 15 min at room temperature. Hearts were then fixed in 4% paraformaldehyde overnight before embedding in OCT compound (Thermo Scientific) with dry ice. In all, 10- µm cross-sections of the heart were made by using HM525 NX Cryostat (Thermo Scientific) starting from the apex to top. The sections were stored in −80 oC before use. The number of capillaries were counted in five random microscopic fields using a fluorescence microscope (Nikon) and expressed as the number of capillaries per square millimeter tissue.
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