Complement IHC ex vivo Sample Clauses

Complement IHC ex vivo. Immunohistochemistry (IHC) was performed on tissue sections. Optimal concentrations of all antibodies used were determined in pilot experiments. IHC staining of biopsies was performed using the peroxidase anti-peroxidase (PAP) method. The PAP method is a further development of the indirect staining method. The indirect technique involves an unlabelled primary antibody (first layer) which reacts with the tissue antigen and a labelled secondary antibody (second layer) which reacts with the primary antibody. The secondary antibody recognises the immunoglobulin of the animal species in which the primary antibody has been raised. This method ensures improved signal amplification through several secondary antibody reactions with different antigenic sites on the primary antibody and thus is a more sensitive method. The second layer antibody can be labelled with a fluorescent dye (indirect immunofluorescence) or with an enzyme such as a peroxidase or alkaline phosphatase. The PAP method involves adding a layer which is an antibody to peroxidase, coupled with peroxidase, making a very stable peroxidase anti-peroxidase complex. This complex acts as a third layer antigen, greatly increasing the sensitivity of the method because the peroxidase molecule is not chemically conjugated to the anti-Ig, but immunologically bound and loses none of its activity. It also allows for a much higher dilution of the primary antibody. The peroxidase is then developed by DAB or other substrates to produce a colorimetric end product. An initial blocking step was performed to block endogenous peroxidase in the tissue using hydrogen peroxide (H2O2; 1:30), made up in a 3 molar solution of sodium nitrite (made in PBS). Bronchial tissue was blocked for 40 minutes and then the slides were washed in PBS twice for 5 minutes each time. The slides were then incubated overnight at room temperature with the control and primary antibodies (Table 2.2), which were made up using 5% normal human serum (in PBS). The slides were washed twice in PBS for 5 minutes each time, and then the monoclonal mouse antibody labelled slides were treated with the second layer rabbit anti-mouse (1:30; DakoCytomation, Inc) in 5 % serum in PBS for 30 minutes whilst the rabbit (anti-C5b-9) slides were treated with the second layer mouse anti-rabbit (1:50; Dako) in 5 % serum in PBS for 30 minutes. After a further wash in PBS for 5 minutes twice, the slides were incubated with the third layer: mouse anti-C3, C3d, C3aR and C5...