Rabbit anti mouse hrp conjugate

For a pilot study, formalin-fixed paraffin-embedded human lung tissues obtained from two SSc-PAH patients were used for immunohistochemistry studies. Briefly, the lung tissues were cut at 3 microns sections, mounted on glass slides, and deparaffinized. For RAGE immunohistochemistry, antigen retrieval was performed with tris-HCl and EDTA. Mouse-anti-human RAGE (Santa Cruz, (A-9): sc-365154, Dallas, Texas, USA) was added overnight. Consecutively, samples were incubated with rabbit anti-mouse-HRP conjugate (Dako, 0260, Santa Clara, USA), and stained with diaminobenzidine as a substrate (Dako K4006, Santa Clara, USA). Negative control staining was performed by omission of RAGE antibody showing no aspecific binding. For determining RAGE localization, additional staining was performed on serial sections as follows. For comparison, standard histochemical Haematoxylin-Eosin and Verhoeff’s elastin stains were performed, in addition to, immunohistochemical stains for CD31 (endothelial cells), alpha-smooth muscle actin ([α-SMA] myofibroblasts and smooth muscle cells), and desmin (smooth muscle cells). The latter immunohistochemical stains were performed using an automated Ventana Benchmark Ultra immunostainer (Roche Diagnostics Netherlands) in our ISO15189 accredited pathology department.

SDS-PAGE was carried out as described elsewhere, in 8%, 10% or 15% SDS-polyacrylamide gels. The gels were either stained with Coomassie blue R250 or, for western analysis, transferred onto PVDF membranes. Blocking was performed in 5% or 10% skimmed milk solution in PBS or PBST (PBS + 0.1% TWEEN-20). Primary antibodies were: mouse monoclonal anti-TAC102 (1:1000, GenScript), rat polyclonal anti-TAC102 (1:1000, Eurogentec), rabbit anti-Protein A (1:5000, Sigma), rabbit anti-myc (1:1000, Sigma), mouse anti-myc (1:1000, Sigma), mouse anti-EF1α (1:10000, SantaCruz), rabbit anti-ALBA3 (1:1000, [53 ]), rabbit anti-ATOM (1:10000, [54 (link)]), mouse anti-GFP (1:1000, Sigma), rabbit anti-GFP (1:1000, Sigma), rabbit anti-COXIV (1:1000), rabbit anti-LipDH (1:10000, [55 (link)]). Secondary antibodies were: mouse anti-rabbit HRP-conjugate (1:10000, Dako), rabbit anti-mouse HRP-conjugate (1:10000, Dako), swine anti-rabbit HRP-conjugate (1:10000, Dako), goat anti-rat 680 LT (1:10000, LI-COR), goat anti-mouse 800 CW (1:10000, LI-COR), goat anti-rabbit 680 LT (1:10000, LI-COR).