Histochemistry of Single Molecules by Carlo Pellicciari & Marco Biggiogera

Author:Carlo Pellicciari & Marco Biggiogera
Language: eng
Format: epub
Publisher: Springer New York, New York, NY

3.Heat the sections in antigen retrieval solution for 15 min at 95 °C.

4.Treat the sections with the blocking solution, antibody to the second target antigen and HRP-labeled antibody. Visualize the antigen localization with the Ni-Co-DAB solution.

Fig. 3Double immunostaining with the HRP-labeled method and immunofluorescence method. (A, B) Localization of platelet and endothelial cell adhesion molecule-1 (PECAM1) and PCNA in the uteri of 10-day-old (A) and 15-day-old (B) mice. Frozen sections were fixed with 10 % formalin /0.1 M phosphate buffer, pH 7.4 for 10 min and treated with anti-PECAM1 rat monoclonal antibody, successively with anti-rat IgG rabbit antibody, and then with Envision plus (rabbit). PECAM1 localization is detected by imidazole-DAB solution (brown). The sections were further fixed with 10 % formalin /0.1 M HEPES buffer, pH 7.4 containing 25 mM CaCl2 for 5 min and heated in 20 mM Tris–HCl, pH 9.0 at 95 °C for 15 min, and then PCNA was localized using mouse monoclonal antibody with Ni-Co-DAB solution (dark purple). Both epithelial cells (outlined arrows) and endothelial cells (arrows) are positive for PCNA-immunostaining on day 10. Endothelial cells show positive PCNA-immunostaining, whereas few epithelial cells reveal PCNA-immunostaining on day 15; blood vessels are continuously formed, whereas the growth of epithelial cells is down-regulated on day 15. (C) Similutaneous staining of clathrin and translocase of mitochondrial outer membrane 20 (TOM20) using immunofluorecence method in the mouse kidney of FFPE specimen. After sections were autoclaved in 20 mM Tris–HCl, pH 9.0 at 121 °C for 10 min, clathrin was detected using mouse monoclonal antibody and Alexa Fluor anti-mouse IgG 549 (red) and TOM20 was localized with rabbit polyclonal antibody and Alexa Fluor anti-rabbit IgG 488 (green). Nuclei were counter-stained with DAPI (blue). Clathrin (arrows) is localized underneath the brush borders which may correspond to apical canaliculi in the proximal tubules. TOM20 is demonstrated in the mitochondria locating in basolateral cytoplasm of the proximal and distal tubules and collecting ducts. (D) Double immunostaining of claudin-5 and β-actin in the glomerulus of mouse kidney in the FFPE tissue. After HIAR in 20 mM Tris–HCl, pH 9.0, β-actin localization was demonstrated with the monoclonal antibody and Alexa Fluor anti-mouse IgG 549 (green) and claudin-5 was localized with rabbit antibody and Alexa Fluor anti-rabbit IgG 488 (red). β-actin and claudin-5 are colocalized along the walls of capillaries (arrows) showing yellow to orange color, which might correspond to pedicels of podocytes. β-actin is also demonstrated in the mesangial cells (outlined arrows). Scale bars: A and B = 50 μm; C and D = 20 μm

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