Antibody incubation at 37°C improves fluorescent immunolabeling in free-floating thick tissue sections
Xia Xiao,
Ya-Ping Feng,
Bin Du,
Han-Ru Sun,
You-Quan Ding,
Jian-Guo Qi
Affiliations
Xia Xiao
1Department of Histology, Embryology and Neurobiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chngdu, Sichuan, China
Ya-Ping Feng
1Department of Histology, Embryology and Neurobiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chngdu, Sichuan, China
Bin Du
1Department of Histology, Embryology and Neurobiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chngdu, Sichuan, China
Han-Ru Sun
1Department of Histology, Embryology and Neurobiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chngdu, Sichuan, China
You-Quan Ding
1Department of Histology, Embryology and Neurobiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chngdu, Sichuan, China
Jian-Guo Qi
1Department of Histology, Embryology and Neurobiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chngdu, Sichuan, China
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.
