Mice were deeply anesthetized and perfused with ice-cold PBS (pH 7.4) followed by 4% paraformaldehyde, PBS (pH 7.4). Brains were promptly dissected and post-fixed in 4% paraformaldehyde, PBS (pH 7.4) for 2 hr on ice. Fixed brains were washed in PBS, and either 250-µm coronal sections were prepared using a vibratome or 25-µm coronal sections were prepared using a cryostat. Brain sections were permeabilized and blocked in permeabilization buffer (3% BSA, 0.3% Triton X-100, and PBS [pH 7.4]) with rocking overnight at 4°C. Primary antibodies were diluted in permeabilization buffer and secondary antibodies were diluted in permeabilization buffer with 5% goat serum. The brain sections were incubated with antibody solutions overnight rocking at 4°C. After antibody incubations, brain sections were washed in PBS (pH 7.4) five times for 30 min with shaking at room temperature. Prior to imaging, floating brain sections were mounted onto glass slides and dried. Fluoro Gel with DABCO (Electron Microscopy Sciences) was applied to each slide and a coverslip mounted on top. A full list of antibodies used, including concentrations and catalog numbers, may be found in the Supplemental Experimental Procedures .
Fluoro gel with dabco
Manufactured by Electron Microscopy Sciences
Sourced in United States
Fluoro Gel with DABCO is a mounting medium designed for the preservation and visualization of fluorescent-labeled samples. It contains the antifade agent DABCO, which helps to maintain the brightness and stability of fluorescent signals. This product is suitable for a variety of fluorescence microscopy applications, but a detailed description of its intended use cannot be provided without the risk of unintentional bias or extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 ab150105, by Abcam (1 mentions)
Birc5, by Cell Signaling Technology (1 mentions)
Las x software, by Leica (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Dfc7000 t camera, by Leica (1 mentions)
Dm2500 microscope, by Leica (1 mentions)
Tcs sp8 system, by Leica (1 mentions)
M o m kit, by Vector Laboratories (1 mentions)
7 protocols using fluoro gel with dabco
1
Immunohistochemical Analysis of Mouse Brains
2
Multicellular Tumor Spheroid Analysis
3
Immunofluorescence Analysis of Cytoskeletal Proteins
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Cells were plated on 12 mm circular coverslips precoated with poly-d -lysine 24 h before fixation with 100% ice-cold methanol for 10 min at –20°C. Cells were washed 2X in phosphate-buffered saline (PBS), permeabilized with 0.25% Triton X-100 in PBS for 10 min, and blocked for 1 h with blocking buffer composed of 10% goat serum in PBS. Cells were stained overnight at 4°C with primary antibodies diluted in blocking buffer at the appropriate dilutions (mouse anti–γ-tubulin [ab11317], 1:500, Abcam; rabbit anti–GSK3-phosphorylated β-catenin [9561], 1:500, Cell Signaling Technologies) followed by 3X wash in PBS and 1 h incubation with appropriate secondary antibodies diluted in blocking buffer (oat anti-rabbit Alexa Fluor 594 [A-11008], 1:500, Thermo Fisher Scientific; and goat anti-mouse Alexa Fluor 488 [ab150105], 1:500, Abcam). After 3X wash with PBS, cells were counterstained with 4′,6-diamidino-2-phenylindole (50 μg/ml), mounted in Fluorogel with DABCO (Electron Microscopy Sciences), sealed with nail polish, and analyzed with a Zeiss D1 epifluorescence microscope with a 63X oil-immersion objective lens (NA = 1.4). Fluorescent images were processed and using ImageJ (National Institutes of Health).
4
Immunofluorescence Imaging of Mouse Brains
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Mice were transcardially perfused with PBS followed by 4% paraformaldehyde in PBS. Brains were dissected and post-fixed with 4% paraformaldehyde, washed with PBS, and embedded in 3% low melting point agarose. Brains were sectioned with a vibratome at the coronal plane, at 150 μm thickness, and processed for immunofluorescence floating in PBS in culture dishes. Slices were incubated with permeabilization solution (PBS, H2O, Bovine Serum Albumin, Triton X-100) at 4°C for at least 6 hr with gentle agitation. Next, slices were incubated with anti-neurofilament (2H3) antibody (mouse monoclonal, Developmental Studies Hybridoma Bank) diluted in permeabilization solution at 4°C overnight. Next, they were washed with PBS at room temperature and subsequently incubated with secondary antibody diluted in permeabilization solution supplemented with 5% normal goat serum at 4°C overnight, washed with PBS, and mounted on microscope slides with PBS. Excess PBS was removed and slices were left to dry for 30 min and subsequently covered with Fluoro Gel with DABCO (Electron Microscopy Sciences, Cat no. 17985) and a rectangular coverslip.
5
Immunohistochemical Analysis of Embryonic and Postnatal Kidneys
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Kidneys dissected from embryonic day 14.5 (E14.5) and postnatal day 0 (P0) mice were fixed overnight in 4% paraformaldehyde, embedded in paraffin and sectioned at 4 μm. After deparaffinization, rehydration, and permeabilization in PBS-Tween (PBS-T), antigen retrieval was performed by boiling the slides in 10 mM sodium citrate pH 6.0 buffer for 30 min. Next, sections were blocked in 3% bovine serum albumin (BSA) and incubated overnight with antibodies recognizing BIRC5 (#2808, Cell Signaling Technology, Danvers, MA, USA), Cyclin D1 (#2978, Cell Signaling) and Neural cell adhesion molecule (C9672, Sigma-Aldrich, St. Louis, MO, USA) at the dilutions recommended by the manufacturers. On the next day, sections were washed with PBS-T, incubated with secondary antibodies at the dilution of 1:200, washed again with PBS-T, and mounted in Fluoro Gel with DABCO (Electron Microscopy Science, Hatfield, PA) before being visualized with a Leica DM2500 microscope and photographed with a Leica DFC 7000 T camera using LAS X software (Leica, Buffalo Grove, IL, USA). Goat anti-rabbit 594 (#111–515-047) and donkey anti-mouse 488 (#715–545-151) antibodies were purchased from Jackson InmmunoResearch Laboratories (West Grove, PA, USA).
6
Immunohistochemical Analysis of Extracellular Matrix
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Tissues sections were first deparaffinized and subjected to antigen retrieval using a citrate buffer at 95 °C for 20 min as previously described [29 (link)]. Immunostaining was performed by using antibodies of Col-1 (1310-01, SouthernBiotech), α-SMA (17H19L35, Thermo Fisher Scientific) and FN1 antibody (MAB1918, R&D Systems). Briefly, mouse tissue sections were blocked using a proprietary blocking solution from a M.O.M. kit (Vector Laboratories, Burlingame, CA, USA). Primary antibodies were then incubated overnight at 4 °C in kit diluent. First antibodies were visualized with Alexa Fluor 488, 568 and 647 secondary antibodies (Life Technologies, Carlsbad, CA, USA), and nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific). Tissues were mounted onto slides with Fluoro Gel with DABCO (Electron Microscopy Sciences, Hatfield, PA, USA). Tissue staining images were taken by Leica TCS SP8 systems (Leica Microsystems, Wetzlar, Germany).
7
Multicellular Tumor Spheroids Formation and Analysis
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