title: Antibody incubation at 37°C improves fluorescent immunolabeling in free-floating thick tissue sections.
abstract: Fluorescent immunolabeling and imaging in free-floating thick (50-60 μm) tissue sections is relatively simple in practice and enables design-based non-biased stereology, or 3-D reconstruction and analysis. This method is widely used for 3-D in situ quantitative biology in many areas of biological research. However, the labeling quality and efficiency of standard protocols for fluorescent immunolabeling of these tissue sections are not always satisfactory. Here, we systematically evaluate the effects of raising the conventional antibody incubation temperatures (4°C or 21°C) to mammalian body temperature (37°C) in these protocols. Our modification significantly enhances the quality (labeling sensitivity, specificity, and homogeneity) and efficiency (antibody concentration and antibody incubation duration) of fluorescent immunolabeling of free-floating thick tissue sections.
authors.affiliation: Department of Histology, Embryology and Neurobiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chngdu, Sichuan, China.
authors.forename: Xia
authors.initials: X
authors.lastname: Xiao
authors.affiliation: Department of Histology, Embryology and Neurobiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chngdu, Sichuan, China.
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Feng
authors.affiliation: Department of Histology, Embryology and Neurobiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chngdu, Sichuan, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Du
authors.affiliation: Department of Histology, Embryology and Neurobiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chngdu, Sichuan, China.
authors.forename: Han-Ru
authors.initials: HR
authors.lastname: Sun
authors.affiliation: Department of Histology, Embryology and Neurobiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chngdu, Sichuan, China.
authors.forename: You-Quan
authors.initials: YQ
authors.lastname: Ding
authors.affiliation: Department of Histology, Embryology and Neurobiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chngdu, Sichuan, China.
authors.forename: Jian-Guo
authors.initials: JG
authors.lastname: Qi
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism*; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006358:Hot Temperature; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D012867:Skin / Q000737:chemistry; D013116:Spinal Cord / Q000737:chemistry
chemical_list: D000906:Antibodies; D005456:Fluorescent Dyes
keywords: fluorescent immunolabeling; free-floating thick tissue section; three-dimensional quantification; whole-mount