Full-thickness biopsies of the grafts were obtained before and after grafting. Tissue sections were incubated overnight at 4° C with the primary antibody (rabbit anti-mouse type I collagen; 1:200; Sigma) [44 (link)]. Tissue sections were washed three times with PBS and then incubated at 37° C for 1 h with biotin-labeled rat anti-rabbit IgG (1:200; Invitrogen) [45 (link)]. Signals were observed using the avidin–biotin–horseradish peroxidase detection system. Slides were examined under an Olympus BX51 microscope.
Immunofluorescence staining was performed at 4° C overnight with guinea pig anti-mouse perilipin (1:400; Progen, Heidelberg, Germany) [46 (link)]. Tissue sections were washed three times with PBS and then incubated at 37° C for 1 h with rhodamine-conjugated goat anti-guinea pig Alexa Fluor 647 IgG (Abcam, Cambridge, MA, USA) [45 (link)]. Nuclei were stained with DAPI (1:200; Sigma). Images were acquired and analyzed under a C1Si confocal laser scanning microscope (Nikon, Tokyo, Japan).
Immunofluorescence staining was performed at 4° C overnight with guinea pig anti-mouse perilipin (1:400; Progen, Heidelberg, Germany) [46 (link)]. Tissue sections were washed three times with PBS and then incubated at 37° C for 1 h with rhodamine-conjugated goat anti-guinea pig Alexa Fluor 647 IgG (Abcam, Cambridge, MA, USA) [45 (link)]. Nuclei were stained with DAPI (1:200; Sigma). Images were acquired and analyzed under a C1Si confocal laser scanning microscope (Nikon, Tokyo, Japan).
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