Diamino benzidine h2o2 substrate

DNA corresponding to deletion constructs of PFA0660w and PfHsp70-x were PCR amplified, and cloned in pET-28a(+) bacterial expression vector before expression in E. coli BL21 (DE3) cells. Recombinant proteins were purified using Ni-NTA affinity chromatography followed by gel permeation chromatography. Gel filtration chromatography was performed using AKTA prime plus on Superdex 200 10/300 GL column (GE Healthcare Life Sciences, Chicago, IL, USA). Purified PFA0660w-S was used for raising polyclonal antibodies in rabbits commercially (Merck, Bangalore, India).
To confirm protein identity, purified recombinant protein constructs of PFA0660w and PfHsp70-x were resolved on SDS-PAGE and transferred on NC membrane. NC membrane was blocked overnight at 4 °C in 5% BSA. Following washing, the blot was probed with horseradish peroxidise (HRP) linked monoclonal anti-hexahistidine antibody (1:2000; Sigma) for 2 hours, and developed with diamino benzidine/H₂O₂ substrate (Sigma).
Recombinant ATS domain of PF08_0141 was purified for experiments as previously described^{36 ,37 (link)}. Briefly, protein was purified using Ni-NTA affinity chromatography followed by Q-Sepharose anion exchange chromatography (GE Healthcare) and then size exclusion on Superdex 200 10/300 GL column (GE Healthcare).

A sterile container was used to collect the CAM membranes, and it was fixed in a 10% formalin solution. For 2 h, CAM paraffin slices (6 μm) were incubated at 60°C on a heat plate. A xylene and ethanol solution was applied to dewaxed membrane sections followed by PBS wash. A heat plate was used for antigen retrieval at 37°C for 10 min, and 1% protease (Sigma-Aldrich, USA) was added. In order to reduce the amount of endogenous peroxidase activity in the sections, 0.3% hydrogen peroxide was used. The samples were then blocked with goat serum for 30 min prior to incubating overnight at 4°C with polyclonal anti-vimentin antibodies (1 : 500), anti-CD34 antibodies (1 : 500), and anti-CD44 antibodies (1 : 500). After this, the sections of membrane were incubated for 1 h at room temperature with goat secondary antibody (anti-mouse 1 : 500 (Dako, USA), followed by peroxidase-HRP conjugated at 1 : 500 (Dako). Diaminobenzidine/H₂O₂ substrate (Sigma-Aldrich, USA) was used to detect the immune reactivity. To counterstain the sections, 10% hematoxylin was applied and the sections were dehydrated and mounted in Pertex (Medite Medizintechnik, Germany).