For the human tissues, 4-μm-thick frozen sections of intestinal specimens were fixed in cold acetone for 10 min at −20°C and blocked with 5% BSA for 1 h at room temperature, then incubated with a primary rabbit anti-human 5-HT2AR antibody (ab66049; rabbit polyclonal to 5-HT2AR; reacts to mouse, rat, human; Abcam, Cambridge, UK; 1:500) overnight at 4°C. Mouse anti-human CD68 antibody (MCA5709; mouse anti-human CD68, monoclonal antibody; AbD Serotec, Kidlington, UK; 1:500) (overnight at 4°C) was subsequently used to detect the macrophages. For the murine colon tissues, 5-HT2AR expression was detected by overnight incubation at 4°C with rabbit anti-mouse 5-HT2AR antibody (ab66049; rabbit polyclonal to 5-HT2AR; reacts to mouse, rat, human; Abcam; 1:300). Subsquently, rat anti-mouse CD68 antibody (MCA1957GA; rat anti-mouse CD68, monoclonal antibody; AbD Serotec; 1:500) was used overnight at 4°C. For both analyses, Alexa-Fluor 488- and Alexa-Fluor 555-conjugated antibodies [goat anti-rabbit IgG (ab150077, goat anti-rat IgG (ab150158) and goat anti-mouse IgG (ab150118); all from Abcam, Cambridge, UK; 1:1,000; 1 h at room temperature] were used as secondary antibodies, followed by incubation with 1 μg/ml DAPI (20 min, room temperature). The sections were finally visualized under a confocal microscope (Olympus, Tokyo, Japan). Images were captured using FluoView software.
Ab66049
Manufactured by Abcam
Sourced in United Kingdom
Ab66049 is a rabbit polyclonal antibody that detects Keratin 14 in a variety of species. It is used for immunohistochemistry and Western blotting applications.
Automatically generated - may contain errors
Lab products found in correlation
Rat anti mouse cd68, by Bio-Rad (1 mentions)
Confocal microscope, by Olympus (1 mentions)
Mca1957ga, by Bio-Rad (1 mentions)
Ab150158, by Abcam (1 mentions)
Ab150118, by Abcam (1 mentions)
Ab150077, by Abcam (1 mentions)
Axio imager a2, by Zeiss (1 mentions)
Rm2135, by Leica (1 mentions)
Ab216880, by Abcam (1 mentions)
Ab85615, by Abcam (1 mentions)
Ab216327, by Abcam (1 mentions)
3 protocols using ab66049
1
Visualizing 5-HT2A Receptor and Macrophages
2
Co-localization of Dopamine and Serotonin Receptors
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To co-localize D2/5-HT1A receptors and 5-HT1A/5-HT2A receptors in the brain, mouse brain was fixed in 4% formaldehyde for overnight, then embedded in paraffin blocks and sectioned using a microtome. To stain D2, 5-HT1A, and 5-HT2A receptors, anti-D2 receptor antibody (H-50, sc-9113, Santa Cruz Biotechnology, Inc.; 1:50 dilution), anti-SR-1A antibody (C-19, sc-1459, Santa Cruz Biotechnology, Inc.; 1:50 dilution), and anti-5-HT2A receptor antibody (ab66049, Abcam; 1:200 dilution) were used, respectively. Dilutions of secondary antibodies (Alexa Fluor 488 Donkey anti-Rabbit IgG, ref. A21206; Alexa Fluor 555 Donkey anti-Goat IgG, ref.A21432; Alexa Fluor 555 Donkey anti-Rabbit IgG, ref. A31572; Invitrogen) were prepared in 5% NDS in concentration 1:100 in all cases. Imaging was done by fluorescence microscope (Zeiss Axio Imager. A2, Poland) using a 20× objective and the following excitation wavelengths: 563 nm for 5-HT1A receptor, 490 nm for D2R and 5-HT2A receptor, and 358 nm for DAPI.
3
Immunohistochemistry of Brain and Colon
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Mice were deeply anesthetized with pentobarbital sodium (40 mg/kg, i.p.), and subsequently perfused with 0.1 M PBS (pH 7.4, 37°C), followed by fixation in 4% (w/v) paraformaldehyde in 0.1 M PBS. The colon and brain tissue samples were sliced into 5 μm sections using a crystal microtome (Leica RM 2135, Wetzlar, Germany), followed by xylene dewaxing and gradient ethanol hydration as described previously. Tissues on slides were equilibrated in 0.1 M Tris-buffered saline solution for a duration of 10 min and blocked with 10% goat serum for 1 h. Subsequently, tissues of hippocampus were incubated with primary antibodies for 1 h, including anti-5HT1A (1:100, ab85615, abcam), or anti-5HT2A (1:100, ab66049, abcam), and tissues of colon were incubated with primary antibody anti-ZO-1 (1:100, ab216880, abcam) or anti-occludin (1:100, ab216327, abcam) for 1 h, respectively. Following primary antibody incubation, the slides were thoroughly washed with PBS and subsequently subjected to secondary antibody incubation. Finally, the slides were mounted with glycerin-based mounting media and examined using a fluorescence microscope.
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