An ABC method (Vector Laboratories) was performed in combination with 3,3'-diaminobenzidine
(Dojindo Laboratories, Japan) for antigen detection.
Endogenous peroxidase activity was blocked using 3% [H.sub.2][O.sub.2] diluted in methanol, reagents were applied manually, and the immunoreactivity was visualized by 3,3'-diaminobenzidine
(Lab Vision DAB substrate system; Lab Vision, Fremont, California, USA).
Antibody-antibody-conjugate complexes were detected with an enzyme substrate comprising 3,3'-Diaminobenzidine
enhancer Abbreviations: Ab, antibody; CC, cell condition; DAB, 3,3'-diaminobenzidine
; GtAMs, goat anti-mouse; GtARb, goat anti-rabbit; HIER, heat-induced epitope retrieval; IgG1, immunoglobulin G1; Phe, phenylalanine; Thr, threonine; TRS, target retrieval solution.
After that, the reaction was visualized using 3,3'-Diaminobenzidine
(K3468, Dako, Glostrup, Denmark) for 2 min.
Pretreatment using a heat-induced epitope retrieval (HIER) procedure prior to IHC was performed and DAB (3,3'-diaminobenzidine
tetrahydrochloride) was used as a chromogen.
Al dia siguiente se efectuo la incubacion con el anticuerpo secundario biotinilado (AM-B1-UNL) en dilucion 1/100 a temperatura ambiente por 30 min y luego exposicion de 30 min a extravidina (Sigma) en dilucion 1/200 y solucion DAB (3,3'-diaminobenzidine
) durante 1 min.
Antigen-antibody complexes were detected by the avidin-biotin-peroxidase method with 3,3'-diaminobenzidine
staining was performed with bond polymer detection kit and ultra view 3,3'-diaminobenzidine
(DAB) detection kit and the sections were developed with diaminobenzidine tetrahydrochloride and counterstained with hematoxylin.
Each slice was then stained with 3,3'-diaminobenzidine
staining, restained with hematoxylin and eosin dehydrated with graded alcohol and mounted using neutral gum.
Ltd.; Beijing, China) for 30 min followed by 3,3'-diaminobenzidine
(DAB) and hematoxylin staining, respectively.
Caption: Figure 1: SERT expression is specific and sensitive to the lung microvasculature (3,3'-diaminobenzidine
, SERT antibody expression in brown).