Antibody incubation at 37°C improves fluorescent immunolabeling in free-floating thick tissue sections

BioTechniques

Bin Du¹  Xia Xiao¹  Ya-Ping Feng¹  You-Quan Ding¹  Jian-Guo Qi¹  Han-Ru Sun¹ 

[1] 1Department of Histology, Embryology and Neurobiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chngdu, Sichuan, China;

关键词: fluorescent immunolabeling;    free-floating thick tissue section;    whole-mount;    three-dimensional quantification;   

DOI  :  10.2144/000114524

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown