Immunofluorescence protocol
SECONDARY, -20
Samples were cut at 7 μm in transverse orientation using a microtome cryostat at -20 °C (Leica CM1850, Wetzlar, Germany). Sections were mounted on superfrost microscope slides (Sigma-Aldrich, catalogue #Z692255), then air-dried for 1 h. Slides were then either stored at -80 °C, or transferred straight into 1 × TBS, pH 7.6 for initiation of a staining protocol. The following antibodies were used for visualization of insulin signalling: 1:250 anti-GLUT4 rabbit polyclonal primary antibody (Abcam, catalogue # ab654) [Tissue samples were blocked for 30 min in 1% bovine serum albumin (BSA), then after washing for 3×5 min in 1 × TBS, samples were incubated in primary antibodies overnight at 4 °C. Next, samples were washed 3×5 min with 1 × TBS, 3×5 min with 2 × TBS and 3×5 min with 1 × TBS. Samples were then incubated with appropriate secondary antibodies, washed thoroughly and subsequently incubated for 1 min in TrueBlack (Lab Supply, NZ, catalogue #23007) to mask tissue autofluorescence. Finally, the samples were washed thoroughly, mounted in 50/50 TBS/glycerol and coverslipped.