Anti cd68 clone ed1

Immunohistochemistry (IHC) for the detection of CD68+ and CD3+ cells in the hernia was performed by the Novolink^TM Polymer Detection Kit (Leica Biosystems, Newcastle, UK) following the manufacturer’s instructions. Antigen retrieval was performed through incubation in near-boiling point 10 mmol/L sodium citrate buffer with a pH of 6.0 for 1 min, followed by incubation with 20 μg/mL proteinase K (Sigma-Aldrich Inc., St Louis, MO, USA) solution for 15 min at 37 °C. Sections were incubated with anti-CD68 (clone ED1, 1:100 dilution, Bio-Rad Laboratories, Irvine, CA, USA) or anti-CD3 (undiluted, Leica Biosystems, Newcastle, UK) antibodies overnight at 4 °C. The stained sections were imaged with light microscopy. CD3+ and CD68+ cells were quantified using ImageJ tools directly on the acquired images. From these, the %CD3 positivity was normalized to the total number of cells in the hernia area, and the %CD68 positivity was normalized to the area of the hernia.

For collagen type II, tissue slices were incubated with the primary antibody anti‐collagen II‐II6B3 (Developmental Studies Hybridoma Bank, Iowa City, IA, http://dshb.biology.uiowa.edu), followed by incubation with the secondary antibody Alexa Fluor 488 goat anti‐mouse (Thermo Fisher). The total number of positive pixels per tissue area was determined as a percentage of fluorescence divided by the total pixels of each image tile, using Interactive Data Language version 8.2 (IDL; Exelis Visual Information Solutions, Inc./Harris Corp., http://www.exelisvis.com). For the following antibodies, the Novolink Max‐Polymer detection system (Leica Biosystems) was used, according to the manufacturer’s instructions: anti‐aggrecan (Santa Cruz Biotechnology, Santa Cruz, CA, https://www3.scbt.com), anti‐fibronectin (Santa Cruz), anti‐Ki‐67 (Thermo Scientific Life Sciences), anti‐MMP3 (Abcam, Cambridge, MA, http://www.abcam.com), anti‐MMP14 (Abcam), anti‐CD68 clone ED1 (Bio‐Rad Laboratories, Hercules, CA, https://www.bio‐rad‐antibodies.com), and anti‐Pax5 (Leica Biosystems).