Immunofluorescent Labeling of Adipose Tissue

We followed a previously reported protocol 100 (link). Fresh adipose tissues were fixed with 4% paraformaldehyde (PFA) overnight at room temperature. Fixed tissues were cut into 2-3 mm³ pieces, blocked with 5% BSA for 1 hour at room temperature, and incubated with FITC anti-mouse F4/80 Antibody (123107, Biolegend, 1:100) or Alexa Fluor 647 anti-mouse F4/80 antibody (123122, Biolegend, 1:100) or Anti-prosaposin (PSAP) antibody (ab180751, Abcam, 1:100) at 4ºC overnight, followed by Cy2 conjugated secondary antibody (ab6940, Abcam, 1:500) at room temperature for 1 hour. Observation of treated fat tissues was performed on TPFM. F4/80 is a major marker of macrophages 101 (link), and prosaposin (PSAP) is a marker of lipofuscin 102 (link), 103 (link). Both F4/80 and PSAP are used to label macrophages.