Guinea pig anti mouse perilipin

The general and histological changes in fat samples were observed and recorded at days 3, 14, 28, and 60 after fat transplantation, and the long-term retention rate was evaluated. Five-micrometer-thick specimens were prepared and stained with hematoxylin and eosin (HE). Images were captured with an Olympus BX51 microscope and Olympus Dp71 digital camera for analysis. The following antibodies were used for immunostaining: guinea pig anti-mouse perilipin as the primary antibody (diluted 1:400; Progen, Heidelberg, Germany) and Alexa fluor 647-labeled goat anti-guinea pig immunoglobulin G (diluted 1:2,000; Abcam) as the secondary antibody. The cell nucleus was labeled with DAPI (diluted 1:200; Sigma, St. Louis, MO, United States). All data were collected and analyzed by two observers.

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).

Tissue sections were immunofluorescently stained with the following primary antibodies: rat anti-mouse Mac2 (1:200; Cedarlane Corp., Burlington, Ontario, Canada), rabbit anti-mouse CD206 (1:300; Abcam, Cambridge, MA, USA), and guinea pig anti-mouse perilipin (1:200; Progen, Heidelberg Germany), After washing, the samples were incubated with donkey anti-rat-555 IgG (1:200; Abcam) and goat anti-rabbit-430 IgG (1:200; Invitrogen, North Ryde, NSW, Australia). Nuclei were stained with DAPI 33342 (Solarbio, D8200, Beijing),and then samples were examined under a light microscope (Nikon Ni-U, Japan). To assess the regeneration of adipose tissue, the preadipocytes^{13 (link)} were counted in 20 randomized fields on each section (200× magnification). To investigate the effect of two injection strategies on the extent of adipose tissue macrophage infiltration, we evaluated the fluorescence staining of M1(red) M2(yellow) macrophages at 100x magnification. A blinded method was adopted in our experiment. The sample for each mark was unknown by the investigator.