Immpact dab chromogen

The methodology for celloidin removal, antigen retrieval and immunohistochemistry has been described previously^{7 ,9 (link)}. In brief: celloidin sections were immersed a saturated solution of sodium hydroxide in 100% ethylic alcohol (EtOH prepared one hour before) diluted 1:3 with EtOH for one hour, 100% EtOH (3 times x 5 min), and distilled water (3 × 5 min). Sections were immersed in heated antigen retrieval solution −100°C- (diluted 1:500 in double-distilled water, Vector antigen unmasking acidic solution, Vector Labs, Burlingame, CA). Sections were allowed to reach room temperature for 30 min, washed with phosphate-buffered saline –PBS- (3× 10 min PBS) and immediately incubated for 8 minutes in a diluted trypsin solution (1:3, Abcam Trypsin Kit) and washed 4×10 minutes in PBS before immunohistochemistry. Sections were incubated for 2 h with a blocking solution containing 0.1 normal horse serum and 0.5% Triton X-100 (Sigma) in PBS. Followed by the incubation with the primary monoclonal antibodies against Na, K-ATPase α1 at 1:1000 dilution in PBS (α1 subunit, Hybridoma Bank), for 48 h at 4 °C in a humid chamber. Secondary antibodies against mouse labelled with HRP (ABC kit, Vector Labs) were used, the antigen-antibody reaction was visualized with diaminobenzidine (ImmPact^™ DAB Chromogen, Vector Labs).