Ab15520

For the immunohistochemical analysis, paraffin-embedded sections were dewaxed, hydrated, and blocked with 5% bovine serum albumin (SW3015, Solarbio) for 30 min. Then, the sections were incubated with the following primary antibodies at 4 °C overnight: rabbit polyclonal anti-rat/human myelin basic protein (MBP) (1:200, 78896; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-rat/human S100 (1:200, ab868; Abcam, Cambridge, UK), mouse monoclonal anti-rat/human GFAP (1:400, G6171; Sigma-Aldrich, St. Louis, MO, USA), rabbit monoclonal anti-human MBP (1:400, ab133620; Abcam, Cambridge, UK), rabbit polyclonal anti-human S100 (1:300, ab15520; Abcam, Cambridge, UK), and mouse monoclonal anti-human GFAP (1:400, ab8975; Abcam, Cambridge, UK). Subsequently, the sections were rinsed with PBS and incubated with horseradish peroxidase conjugated goat anti-mouse or anti-rabbit secondary antibodies for another 2 h at 37 °C. Finally, the sections were stained with a DAB (3,3′-diaminoben zidine) Kit (Zsbio, Beijing, China) and observed by fluorescence microscope (Axiovert A1, Carl Zeiss, Germany). Areas of positive staining were determined using the Image J software.

Immediately after harvesting the specimens were fixed in 4% buffered formalin solution and embedded in paraffin. The number of sections to be made in each plane of the specimens was based on the size of the mechanoreceptors: Since a single receptor can extend over some 7 μm cuts, the distance between the cut planes was set to 70 μm. This procedure allows the number of receptors counted more than once to be kept very low [12 (link)]. One plane therefore consists of 10 sections; a total of 6 planes (A–F) were cut per sample as shown in Fig. 1.
Fig. 1

Sketch of the sectional planes A–F of one specimen. One plane equals 70 μm

In each of the planes, the first specimen was stained with HE, the second one with polyclonal antibody against S100 (abcam, ab15520), the third one with polyclonal antibody against p75 (sigmaaldrich, N3908) and the fourth one with monoclonal antibody against PGP9.5 (abcam, ab8189). These three antibodies are widely used in immunohistochemical analysis of neuronal structures and have proven to be the most reliant method in the detection of mechanoreceptors [11 (link), 13 (link)–15 ].