Objective To develop a method for immunofluorescent staining in thick skin sections and to enable the thick skin sections to be imaged by a wide-field fluorescence microscope. Methods Five male adult SD rats were perfused with Zamboni’s fixative. The plantar area skin of hindpaws was obtained from the fixed rat. Cryotome sections were cut with 40μm in thickness. The sections were randomly chosen for pan axonal biomarker PGP 9.5 immunostaining. Firstly, effect of optical clearing was evaluated by comparing imaging depths of skin sections with or without gradient glycerol treatment after staining. Secondly, other skin sections were incubated with dimethyl sulphoxide (DMSO) for 10min, 15min and 30min respectively before staining, with glycerol treatment after staining, in order to assess if DMSO deceases background staining and determine the optimized time. To investigate whether morphologic changes occur after gradient glycerol clearing, other two groups of skin sections were selected, treated with glycerol or PBST and observed under an operating microscope. Finally, by utilization our modified method, i.e., a combination of DMSO and gradient glycerol treatment, other skin sections were stained with two nerve fiber biomarker NF 200 and Peripherin respectively. Results Imaging depths of skin sections after gradient glycerol treatment were significantly deeper than those without glycerol clearing (P<0.01). Treating skin with DMSO drastically decreased the background staining of skin sections and 15 minutes incubation of DMSO was the most appropriate. There was no obvious morphologic change in skin sections after gradient glycerol clearing. With our modified method, NF 200-IR and peripherin-IR fibers were effectively labeled. Conclusion By using our modified method, fluorescent immunoreactivities in the thick skin sections can be imaged effectively under a wide-field fluorescent microscope.