Ab8189

Human endometriosis lesion tissue samples were obtained from provitro GmbH (Berlin, Germany). Patients provided informed consent for the provision of all tissue samples. Tissue samples were paraffin embedded, sectioned, and mounted on Superfrost slides. Prior to staining, mounted tissue sections were deparaffinized and rehydrated. Sections were then permeabilized in 0.3% Triton X-100/phosphate buffered saline (PBS) and blocked in 10% normal goat serum/PBS. Sections were then co-stained by incubating with the primary antibodies, α-P2X3 rabbit polyclonal (Abcam, ab10269; 1:1000) and α-PGP9.5 mouse monoclonal (Abcam, ab8189; 1:1000) diluted in 1% normal goat serum and 0.1% Triton X-100 in PBS at 4 °C overnight. Sections were washed three times in PBS for 15 min and incubated with appropriate Alexa Fluor secondary antibodies diluted in 3% normal goat serum for 2 h at room temperature. Sections were washed in PBS as described above and counterstained using DAPI to visualize nuclei. Mounted sections were dried overnight and coverslipped using Flouromount-G. Images were acquired with an Olympus IX81 microscope (10× objective, NA: 0.30, resolution: 0.92 μm, Olympus).

To examine the effect of CEC‐sEVs and oxaliplatin on DRG neurons and IENF, immunofluorescent staining was performed on DRG (8 μm thick) and footpad tissue (20 μm thick) sections, respectively, according to our published protocols (Zhang et al., 1999, 2010, 2013, 2014). The following antibodies were employed: polyclonal anti NF200 (N4142, MilliporeSigma), CGRP (ab43873, abcam) and monoclonal anti‐PGP9.5 (ab8189, Abcam). Immunoreactive cells and nerve fibres were imaged using laser scanning confocal microscopy (LSCM) and were quantified according to our published protocols (Buller et al., 2010; Wang et al., 2014).
To identify the morphometric changes within sciatic nerves, toluidine blue staining was performed on semi‐thin transverse sections (2 μm thick) of sciatic nerve and imaged using a 100x oil immersion lens (Olympus). Myelin sheath thickness, myelinated fibre and axon diameter were measured using the MCID system, as described in our published protocols (Zhang et al., 1999, 2010, 2013, 2014). The myelin sheath and axons in ultra‐structural level were also analyzed by means of TEM.

Light antigen retrieval was performed in 0.1 M of PB with pH 7.3, 0.2% of hydrogen peroxide, and 0.05% of Triton X-100 for 25 min at room temperature (RT). Sections were then washed twice in PBS and incubated in a blocking solution of tris-buffered saline (TBS) with 0.05% of Tween 20, 2% of bovine serum albumin (BSA), and 2% of normal donkey serum (NDS) for 1 h before applying primary antibodies overnight at 4°C. The following primary antibodies (diluted in the blocking solution) were used: Rabbit anti-SCN9A-ATTO Fluor-663 antibody (1:100, ASC-008-FR, Alomone Labs, Jerusalem, Israel), Mouse anti-PGP9.5 antibody (1:200, ab8189, Abcam, Cambridge, United Kingdom), and rabbit anti-PGP9.5 (1:500, ab108986, Abcam). For the detection of the primary antibody, secondary antibody raised in donkey and conjugated with Alexa-647 fluorophore was used (1:500, Molecular Probes, Fisher Scientific, Illkirch, France) for 1 h at RT. DAPI staining (1:2000, Molecular Probes, Thermofisher, Illkirch, France) was performed at the same time as in the secondary antibody. Sections were then washed twice with PBS then placed on superfrost glass slides. One drop of Immu-Mount (Fisher Scientific, Illkirch, France) was added over the tissue sections and a coverglass was placed over the slide.

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 ].