Three-month old Tat+ and Tat− mice, both female and male, were perfused with 4% paraformaldehyde after they were fed with DOX-containing chow for 10 days. The perfused brain was dissected out and fixed with 4% paraformaldehyde overnight before washing three times in PBS and immersed in 20% sucrose solution for 24 hr. The brains were then frozen in OCT compound (Sakura Finetek, Torrance, CA), coronally sectioned at 20 µm thickness, and thaw-mounted onto Superfrost-plus slides (Fisher). The sections were then immersed in acetone at −20℃ for 20 min before they were washed with PBS and blocked with PBS containing 0.5% Triton and 5% cold fish gelatin (Electron Microscopy Science, Hatfield, PA) for 15 min at room temperature and probed with Rb anti-ATX (1:250, Cosmo Bio, Japan) and Ms anti-APC (1:100, Millipore) overnight at 4℃. Images were taken on a confocal microscope (Zeiss LSM 700, Carl Zeiss, Thornwood, NY) and processed using Zeiss Zen 2010 software.
Fish gelatin
Manufactured by Electron Microscopy Sciences
Sourced in United States
Fish gelatin is a natural, water-soluble protein derived from the collagen of fish skin and bones. It is a versatile laboratory material commonly used in various applications, such as tissue culture, histology, and electron microscopy. Fish gelatin provides a non-toxic, gelling, and adhesive agent for stabilizing and embedding samples for microscopic examination.
Automatically generated - may contain errors
Lab products found in correlation
Silver enhancement kit, by Electron Microscopy Sciences (2 mentions)
Lsm 700, by Zeiss (1 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Fish gelatin, by Merck Group (1 mentions)
Formaldehyde, by Ted Pella (1 mentions)
Libra 120 electron microscope, by Zeiss (1 mentions)
Lr white resin, by Polysciences (1 mentions)
Tween 20, by Merck Group (1 mentions)
Nh4cl, by Merck Group (1 mentions)
4 protocols using fish gelatin
1
Visualizing Tat Protein Expression in Mice
2
Ultrastructural Mapping of TDP-43 and NUP98 in Mouse Brain
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Mouse brain tissues for IEM analysis were fixed with 4% PFA overnight. Following the fixation, 40 μm-thick free-floating sections were incubated with a cryoprotectant solution for 2 hours at room temperature and freeze-thawed. The sections were incubated with 4% blocking normal goat serum for 1 hour and with anti-TDP-43 antibody (Proteintech, 10782-2-AP, 1:50) and NUP98 antibody (Abcam, ab50610, 1:50) in 1% blocking normal goat serum overnight. After that, the sections were incubated with 0.2% BSAc and 0.2% fish gelatin (both from Electron Microscopy Sciences, Hatfield, PA) in PBS for 10 minutes and with gold nanoparticle-conjugated secondary antibodies, including goat anti-rabbit IgG (H+L), Ultra Small (Electron Microscopy Sciences, 25101, 1:100) or goat anti-rat IgG (H+L), Ultra Small (Electron Microscopy Sciences, 25181, 1:100) for 2 hours. After the PBS washes, the sections were fixed in 1% glutaraldehyde for 10 minutes. The immunogold particle signal was increased using a silver enhancement kit (Electron Microscopy Sciences) at 37°C for 2 hours. Finally, the sections were post-fixed in 0.5% osmium tetroxide for 10 minutes, dehydrated, and embedded in resin. To quantify TDP-43 granule size, we used NIH ImageJ and set threshold from 0 to 60 using 8-bit images, and run particle analysis to extract granule size and number.
3
Ultrastructural Mapping of TDP-43 and NUP98 in Mouse Brain
4
Ultrastructural Immunostaining of Oocytes
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Oocyte fixation and embedding for electron microscopy were performed using a routine technique [4 (link)]. Oocytes were prefixed for 1.5 h in a solution containing 4 % formaldehyde (Ted Pella, Redding, Calif., USA) and 0.5 % glutaraldehyde in PBS, then fixed overnight in 2 % formaldehyde at 4 °C. After rinsing in PBS containing 0.05 M NH4Cl (Sigma) and subsequent dehydration in an ethanol series, oocytes were embedded in medium grade LR White resin (Polyscience, Warrington, Pa., USA). Ultrathin sections were incubated for 10 min in a blocking buffer containing 0.5 % fish gelatin (Sigma) and 0.02 % Tween-20 (Sigma) in PBS (pH 7.4). Sections were then incubated in the primary antibody solution overnight in a moist chamber at 4 °C. After rinsing in PBS containing 0.1 % fish gelatin and 0.05 % Tween-20, the sections were incubated with secondary goat anti-mouse and goat anti-rabbit IgGs conjugated with 10 nm gold particles (Electron Microscopy Sciences, USA). As a control, additional sections were incubated only in secondary antibodies. Ultrathin sections were contrasted with 1 % uranyl acetate-water solution and examined in a Carl Zeiss Libra 120 electron microscope operated at 80 kV. Magnification was inserted to the images automatically. The figures were prepared in Adobe Photoshop (Adobe Systems).
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