Biotinylated goat anti rabbit ab

For labeling fibroblast-like cells, the polyclonal antibody antiS100a4 ab27957 (Abcam, Cambridge, UK) was used.
Transwell cultures were embedded after 10 and 21 days in Tissue-Tek (Sakura, Zoeterwoude, The Netherlands) and frozen at − 20 °C. Samples were cut into 10 µm serial-sections, mounted on superfrost glass slides (Menzel, Braunschweig, Germany), fixed in acetone, and dried at room temperature. After re-hydrating with 0.2% Tween (Merck, Darmstadt, Germany) in PBS sections were incubated for 1 h at RT with ABO-Serum (Biotest, Dreieich, Germany) mixed with horse serum (both 1:20 in PBS with 3% Bovine Serum Albumin (BSA; Sigma-Aldrich Co.) to block unspecific binding sites. Incubation with the first antibody (AB) anti-S100A4 (Abcam) (1:100 in AB dilution buffer (DCS Innovative, Hamburg, Germany)) for 1 h at RT was performed. After washing, incubation with the biotinylated goat anti-rabbit AB (Vector Laboratories, Burlingame, USA) (1:200 dilution with AB dilution solution) was performed for 30 min at RT. After washing, the sections were treated with avidin–biotin–peroxidase complex (ABC; Vector Laboratories) for 30 min at RT. Then, the sections were washed. Staining was developed using 3-Amino-9-Ethyl Carbazol (Sigma-Aldrich Co.) and counterstained with hemalaun. The sections were then embedded in Aquamount (Merck, Darmstadt, Germany).

Mice were euthanized with CO₂ and hind limbs at the ankle were collected and were fixed in Zamboni fixative (Newcomers Supply, Middleton, WI) for 24–72 hours. Footpads were dissected out and were washed in phosphate buffer and placed in cryoprotectant (30% glycerol) solution. Tissue blocks were cut by freezing microtome at 50 μm intervals and immunohistochemical staining was performed using a standard chromogen technique with the following antibodies: rabbit anti-PGP 9.5 (AbD Serotec, a Bio-Rad Company, Kidlington, UK) and rabbit anti-TH (Novus Biologicals, Littleton, CO). For each marker, four 50 micron sections were selected from footpads 3 and 4. These were selected at random from throughout the possible sections. Sections were incubated over night at room temperature in 96 well tissue culture plates on a horizontal tabletop shaker at 50 rolls per minute. The following day, sections were washed in phosphate buffer 2–3 times and then incubated with biotinylated goat anti-rabbit Ab (Vector Labs, Burlingame, CA) for 2–3 hr. Bound immunoglobulin was visualized by the ABC kit (Vector labs, Burlinggame, CA).