IGF-1R, GFAP and myelin basic protein (MBP) immunofluorescence stainings were performed on cryostat-cut decalcified spinal cord sections as follows: for IGF-1R and GFAP stainings, the tissue was fixed with 100% ice cold acetone at −20 °C for 10 min and dried before being reconstituted in homemade 1xTris-Buffered Saline (TBS). Alternatively, for MBP staining, sections were fixed with 100% ice cold methanol at −20 °C for 10 min and immediately washed for a total of 30 min in TBS. Unspecific antibody binding was blocked for 2 h at room temperature (RT) either with 10% goat serum containing 0.1% Triton (Sigma-Aldrich, St. Louis, MO, USA) diluted in TBS (for IGF-1R and GFAP stainings) or with 5% BSA containing 0.3% Triton diluted in TBS (for MBP staining). Sections were then incubated at 4 °C overnight with the following primary antibodies: polyclonal rabbit anti-IGF1R (phospho-Y1161, Abcam, ab39398, 1:100), polyclonal rabbit anti-GFAP (Dako, Z0334, 1:100) diluted in 2% goat serum in TBS containing 0.1% Triton and a monoclonal rat anti-MBP (aa82-87, BioRad, MCA409S, 1:100) diluted in 1% BSA in TBS containing 0.3% Triton. After rinsing 3 × 10 min with TBS, spinal cord sections were incubated for 2 h at RT with the following secondary antibodies: Alexa Fluor 647 goat anti-rabbit (Invitrogen, A32733), Alexa Fluor 488 goat anti-rabbit (Invitrogen, A11008) diluted (1:200) in 2% goat serum in TBS and Alexa Fluor 488 goat anti-rat (Invitrogen, A-11006) (1:200) diluted in 1% BSA in TBS. Following a 3 × 10 min wash with TBS, slices were incubated with DAPI (1:5000 in TBS, 1 mg/ml stock, AppliChem, Darmstadt, Germany) for 10 min at RT, after which they were mounted with Mowiol 4–88 solution (Sigma-Aldrich, St Louis, MO, USA) and left to dry.
For IGF1-R staining, Z-stack images of CNS sections were acquired using a LSM800 confocal microscope (Zeiss) with 25 × and 40 × objectives. Images of GFAP and MBP stainings were acquired using a fluorescence microscope Nikon Eclipse E600 with 10 × and 20 × objectives. All images were analyzed using the software Fiji (National Institute of Health, Bethesda, MD, USA).
For IGF1-R staining, Z-stack images of CNS sections were acquired using a LSM800 confocal microscope (Zeiss) with 25 × and 40 × objectives. Images of GFAP and MBP stainings were acquired using a fluorescence microscope Nikon Eclipse E600 with 10 × and 20 × objectives. All images were analyzed using the software Fiji (National Institute of Health, Bethesda, MD, USA).
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