Organoids grown in Matrigel were fixed in 4% paraformaldehyde for 10 min, rinsed three times in PBS for 10 min, embedded in Optimal Cutting Temperature compound (OCT), and frozen at −80°C. OCT blocks were sectioned at 50-μm thickness by cryostat at −20°C. Sections were placed on Superfrost Plus Gold microscope slides (15-188-48; Fisherbrand) and stored at −80°C. For antibody staining, samples were thawed at room temperature, rinsed twice in PBS for 10 min to remove OCT, permeabilized with 0.5% Triton X-100 for 1 h, and rinsed twice in PBS for 10 min. Samples were blocked for 1-3 h with 10% FBS/1% BSA, incubated with primary antibodies overnight at 4°C in 1% FBS/1% BSA, and rinsed three times in 1% FBS/1% BSA for 15 min. Incubation with secondary antibodies was conducted in 1% FBS/1% BSA overnight at 4°C. Slides were rinsed three times in PBS for 10 min, mounted with Fluoromount (F4680; Sigma-Aldrich), and sealed with coverslips. Primary antibodies used were mouse anti-Twist1 (1:50; sc-81417; Santa Cruz Biotechnology Inc.) and mouse anti-smooth muscle α-actin (1:250; A5228; Sigma-Aldrich). Secondary antibodies used were all Alexa Fluor-conjugated antibodies (1:200; Invitrogen).
Superfrost plus gold microscope slides
Manufactured by Thermo Fisher Scientific
Sourced in United States
Superfrost Plus Gold microscope slides are designed for a variety of microscopy applications. They feature a gold-colored surface coating that enhances slide visibility and handling.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti α smooth muscle actin, by Merck Group (1 mentions)
Mouse anti twist1, by Santa Cruz Biotechnology (1 mentions)
Fluoromount, by Merck Group (1 mentions)
Prolong diamond antifade reagent, by Thermo Fisher Scientific (1 mentions)
Ab30788, by Abcam (1 mentions)
Goat anti rat igg conjugated to cy3, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Te200 microscope, by Nikon (1 mentions)
Bx51 fluorescence microscope, by Olympus (1 mentions)
Rnascope multiplex fluorescent detection reagents v2, by Advanced Cell Diagnostics (1 mentions)
6 protocols using superfrost plus gold microscope slides
1
Immunofluorescence Staining of Organoid Sections
2
Carotid Artery Cryosectioning and Imaging
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Perfused carotid arteries were sectioned into 5-mm segments and frozen in optical cutting temperature (O.C.T.) compound (Sakura Finetek USA, Torrance, CA, USA). 12-micron cross-sections were cut using a cryostat. Sections were mounted on SuperFrost Plus Gold microscope slides (Fisher Scientific) using Prolong Diamond Anti-Fade Reagent (Invitrogen, Carlsbad, CA, USA) after washing with PBS. Sections were imaged with an Echo Revolve Fluorescence Microscope (Discover Echo, a BICO Company, San Diego, CA, USA) with a FITC filter to detect Flutax-1 fluorescence. Depth of drug penetration was measured from the internal elastic lamina to the maximum penetration depth and normalized to the thickness of the medial layer or wall thickness.
3
Somatostatin Immunohistochemistry in DRGs
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Pieces of fresh frozen DRGs were placed in Tissue-Tek (Sakura, 4583) and sectioned to 10-14 µm thickness. Sections were placed onto Superfrost Plus Gold Microscope slides (Fisher, 15-188-48). Following sectioning, slides were placed in a 4% PFA (Sigma, St. Louis, MO, USA, P6148) for 10 min, washed with 1X PBS 3 times, and then stored at room temperature for 60 min with a 5% BSA (VWR, 0332-100G; w/v), 0.1% Triton-X100 (Sigma, T8787; v/v) blocking solution. The slides were then washed in 1X PBS 3 more times and then stored overnight at 4°C with the primary antibody in a 5% BSA, 0.1% Triton-X100 solution. The following day, slides were washed 3 times in 1X PBS and then incubated at room temperature for 60 min in a 2% BSA, 0.01% Triton-X100 solution with the secondary antibody. Slides were then washed 3 times in 1X PBS, vacuum dried, and mounted with Prolong Diamond Antifade mountant with DAPI (Thermofisher, P36962). Slides were stored at 4°C in the dark until imaging on a Nikon TE200 microscope. The following primary antibody was used to stain for SST: rat anti-human SST (abcam, ab30788; 1:500, which was selected because human and dog SST are 98% identical according to an alignment run using NCBI's BLASTp program. The following secondary antibody was used at a 1:200 dilution goat anti-rat IgG conjugated to Cy3 (Life Technologies, Waltham, MA, USA, A10522).
4
Tissue Fixation and Cryosectioning Protocol
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Study mice were anesthetized with isoflurane followed by transcardial perfusion with heparin (20 U/mL) in neutral phosphate buffer (PB) and then 4% paraformaldehyde (PFA) in PB. Tissue was harvested and placed in ice-cold 4%PFA/PB and stored for 24 hours at 4°C. Samples were then transferred to a 30% sucrose/PB solution for storage at 4°C for 72 hours. Tissue was then dried and frozen rapidly in optimal cutting temperature (OCT) media on a block of dry ice. Samples were then wrapped in aluminum foil, placed in freezer bags and stored at –80°C until sectioning. Tissues were equilibrated to –20°C and then sectioned using a cryostat (model CM1950; Leica, Buffalo Grove, IL, USA) at a thickness of 15 µm directly onto Superfrost Plus Gold microscope slides (Thermo Fisher, Waltham, MA, USA). Slides were dried in a dark drawer overnight. The next day, slides were processed for labeling or placed in a slide holder, sealed, and stored at –80°C.
5
Fluorescence Imaging of nHA-Loaded Scaffolds
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The different nHA loadings were analysed qualitatively by imaging the scaffolds with an Olympus BX51 fluorescence microscope (Olympus Corporation, Tokyo, Japan). The scaffolds were embedded in the Epredia TM M1 embedding matrix for 3 days. Further, the scaffolds were cryo-sectioned at –25 °C at 7 µm thickness with a CM1900 UV Cryostat apparatus (Leica Biosystems). The sections were allowed to adhere to Superfrost Plus® Gold microscope slides (ThermoFisher Scientific, Waltham, MA, USA) overnight prior to staining. A solution of 0.5% calcein in 0.1N NaOH was used to stain Ca2+. The sections were rinsed twice with dH2O to remove the excess stain and left to dry prior to imaging.
6
Detecting Fkn/CX3CL1 mRNA in Phrenic Motor Neurons
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Detection of Fkn/CX3CL1 mRNA using in situ hybridization was done to verify siRNA knockdown in phrenic motor neurons. On day 14, rats were anaesthetized under isoflurane anaesthesia (4% in O2) and transcardially perfused with ice-cold RNase-free 1X phosphate buffered solution (PBS) followed by 4% paraformaldehyde. Spinal segments C3-C5 were harvested and dehydrated in RNase-free 30% sucrose. Twenty micrometer sections were cut on a microtome and collected in 10–15 sets of serial C3-C5 sections in RNase-free PBS, in order to obtain representative staining across segments C3-C5. Sections were mounted on to Superfrost Plus Gold microscope slides (ThermoFisher Scientific, Inc., Waltham, MA). After drying at room temperature for ~2 hours, slide-mounted tissue was stored at −80°C until use. All reagents used for in situ hybridization were purchased from Advanced Cell Diagnostics (Newark, CA). In situ hybridization was used for Fkn/CX3CL1 mRNA detection (reference no. 531141) using a fluorescence-based assay (reference no. 323110), as previously described89 (link). Experimental protocols were performed according to the manufacturer instructions in RNAscope Multiplex Fluorescent Detection Reagents V2 (Document No. UM 323100, Advanced Cell Diagnostics).
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