Presence of TG activity, FXIII-A, and TG2 in mouse WAT and differentiating 3T3-L1 preadipocytes. (A) Immunohistochemical visualization of γ-glutamyl-ε-lysyl bonds (isopeptide bonds) in WAT showing abundant staining in the extracellular compartment. Epididymal fat pad tissue was obtained from 8-week-old mice. Specimens were counterstained with hematoxylin (H) (n = 2). Scale bar represents 100 µm. (B) In vitro TG activity of protein extracts of different WAT fat deposits of 6- to 8-week-old male mice. Protein extracts of epididymal (Ep), mesenteric (Me), perirenal/retroperitoneal (Re), inguinal (In), and subcutaneous (Sc) fat pads were assessed by microplate BPA incorporation assay (n = 2). (C) Reverse transcription-polymerase chain reaction analysis of TG enzyme family members in mouse WAT and 3T3-L1 preadipocytes. Only Tgm2 and F13a1 are expressed (n = 3). (D) Whole-mount immunofluorescence staining of mouse WAT showing the presence of TG2 (red) and FXIII-A (green) in the tissue. Epididymal fat pads of 2 mice were used. Scale bar represents 10 µm. (E) Reverse transcription-polymerase chain reaction analyses of Tgm2 and F13a1 during 3T3-L1 preadipocyte differentiation to adipocytes, showing different expression patterns for the 2 TGs during adipogenesis. Tgm2 levels remain constant while F13a1 levels are high at the early phase of adipogenesis, followed by a gradual decrease as the cells mature to adipocytes. Pparg2 and Cbpa are adipocyte differentiation markers. Gapdh was used as internal control. n = 3.