Biotin labelled secondary antibody

Capillary density was assessed as described previously (Madsen and Holmskov, 1995 (link)). In short, sections were air dried for 10 minutes and fixed in 4% formaldehyde in phosphate buffered saline (PBS). Sections were incubated overnight with primary anti-collagen type IV antibody (1:50 Santa-Cruz, USA), followed by a 1 hour incubation with biotin-labelled secondary antibody (1:100, Vector Laboratories, USA). Subsequently, sections were incubated for 30 minutes in avidin–biotin complex, followed by 10 minutes with 3,30-diaminobenzidine staining. The number of capillaries was determined by visual inspection using a Leica DMRB microscope (Wetzlar, Germany) and a ×40 objective at 436 nm (for details, see Madsen and Holmskov, 1995 (link)).

Samples were cut into 7 μm thin slices, and cryo-sections were fixed in acetone. After blocking in 4% appropriate serum, sections were incubated with primary antibodies (Mac3 (1:200, M3/84, BD Pharmingen), PARP14 (1:50, HPA01206, Sigma-Aldrich), PARP9 (1:100, ab53796, Abcam) and human CD68 (1:200, M0876, Dako), followed by biotin-labelled secondary antibody (1:250, Vector Laboratories, Burlingame, CA, USA) and streptavidin-coupled Alexa Fluor 488 antibody (Life Technologies). For immunofluorescence double labelling, after avidin/biotin blocking (Vector Laboratories), the second primary antibody was applied overnight at 4 °C, followed by biotin-labelled secondary antibody and streptavidin-coupled Alexa Fluor 594 antibody (1:250, Life Technologies). Sections were washed in PBS and embedded in mounting medium containing 4,6-diamidino-2-phenylindole (Vector Laboratories). For bright-field immunohistochemistry on tissue sections, following the first biotin-labelled secondary antibody incubation, sections were incubated with streptavidin-labelled horseradish peroxidase (HRP) solution (Dako), followed by 3-amino-9-ethylcarbazole (AEC) solution. Slides were examined using the Eclipse 80i microscope (Nikon, Melville, NY, USA) or the confocal microscope A1 (Nikon). All images were processed with the Elements 3.20 software (Nikon).