Ab49701

Testicular biopsies, embedded in paraffin, were identical to the ones described in previous studies and were processed for immunohistochemical studies as mentioned4 (link). The local Ethical Committee (Ethikkommission, Technische Universität München, Fakultät für Medizin, München, project number 5158/11) has approved the study. A primary antibody against BGN (1:200; polyclonal affinity isolated rabbit anti-human BGN; #ab49701, Abcam, Cambridge, UK) was applied for Western blotting and immunohistochemistry. A further rabbit antiserum to BGN, used for immunohistochemistry, was bought from Sigma (Deisenhofen; Germany, #HPA003157; 1:500). Rabbit anti-collagen type 1 for immunohistochemistry was from Acris (Herford, Germany #R1038; 1:1000), as was rabbit anti-TLR2 (ARP30551; 1:400). Mouse anti-smooth muscle actin (SMA) for immunohistochemistry stems from from Sigma (AS228; 1:1000). Negative controls included incubation with the corresponding IgG isotype instead of the antiserum, omission of the antiserum, and use of normal serum instead of the antiserum. The BGN antiserum (#ab49701, Abcam) was pre-adsorbed with recombinant BGN and used as a further control for immunohistochemistry. In some cases, sections were counterstained with haematoxylin.

FFPE tissue specimens (included in tissue microarrays) were cut at 3μm thickness, deparaffinized and subjected to antigen retrieval in pH 6.1 citrate-buffer. Subsequently, immunohistochemical staining was done by incubation with the primary anti-Bgn antibody (Abcam, ab49701) or anti-integrin-β1 antibody (clone: EP1041Y, Abcam, ab52971). Bgn and integrin-β1 expression was evaluated in each of 3 separate high power fields (HPF) with a HPF being a 40x magnification. Slides were scored for Bgn and integrin-β1 intensity as follows: score 0 = negative or weak positive expression, score 1 = moderately positive expression and score 2 = strongly positive expression.

Immunofluorescent staining was performed to characterize matrisome protein expression and confirm proteomic profiling results. Frozen sections were used at 3 μm thickness. CryoJane tape was employed to transfer the section to the slide, which were then fixed in paraformaldehyde 4% and washed 2 × 5 min in PBST (phosphate buffered saline with 1% triton X-100). Cryosections where stained with Annexin-A1 (Anxa1, Abcam ab214486, 1:500), Asporin (Aspn, Abcam ab58741, 1:500), Biglycan (Bgn, Abcam ab49701, 1:50), Collagen A1 (III) (Col3a1, Abcam ab6310, 1:200), Lumican (Lum, Abcam ab168348, 1:200), Periostin (Postn, Abcam ab14041, 1:200), Protein S100-A6 (s100a6, Abcam ab244301, 1:50), and Protein S100-A10 (s100a10, Abcam ab187201, 1:50). Primary antibodies were incubated for 2 h, followed by secondary antibodies Alexa Fluor 488 (goat anti-mouse, Invitrogen A21121), Alexa Fluor 488 (goat anti-rabbit, Invitrogen A11008) and Alexa Fluor 555 (goat anti-rabbit, Invitrogen A21422) during 1 h. Stained samples were mounted with Vectashield^® (Vector Laboratories H-1500). Imaging was performed with the LSM 700 confocal laser scanning microscope (Zeiss) at 1 Airy Unit (AU) with wavelengths 416, 485, and 555 nm at 200x magnification.