Ultramount

Dissected skeletal muscles were frozen in isopentane, cooled down with liquid nitrogen, transferred to − 80 °C, and cut into 10 μm sections using cryomicrotome (Microm HM, Thermo Fisher Scientific). Cryosections were fixed with 3% PFA, washed with PBS, and stored in 4 °C. Samples were hydrated in PBS, incubated in Harris hematoxylin solution (Sigma-Aldrich), and washed in distilled water. Then, fixed sections were incubated in eosin Y solution (Sigma-Aldrich) and washed in distilled water. Specimens were mounted with UltraMount (Dako Cytomation) and analyzed using inverted light microscope Eclipse TE200 (Nikon) and ImageJ software (NIH).

In situ hybridization was performed using either the 1-plex Quantigene View RNA ISH Tissue assay kit (Affymetrix, #QVT0051) or the 2-plex Quantigene View RNA ISH Tissue Assay kit (Affymetrix, #QVT0012). Assays were performed as per the manufacturer’s instructions with stock solutions. Before beginning the hybridization protocol, 5um thickness tissue section were baked at 60°C for 30 min to increase tissue adhesion. The paraffin was removed from the slides with xylene before being boiled in a pretreatment solution (Affymetrix) for 10 min and incubated with Protein Kinase K (Affymetrix) at 40°C for 10 min. Custom probes for Human JAK2 and Mouse Jak2 (Affymetrix) were then hybridized to the tissue. Signal amplification was accomplished by hybridizing Type1 and Type2 specific pre-amp oligonucleotides, amp oligonucleotides and labeled oligonucleotides sequentially. Slides were then mounted with an aqueous mounting medium (Dako, Ultra mount). Digital slide image data was generated from the glass slides using the Aperio AT2 slide scanner (Leica Biosystems) for bright field, and the P250 Scanner (3D Histech) for fluorescent acquisition.

ViewRNA Tissue Assay Kit (Affymetrix) and probes specific for IGF-1 mRNA (VB1-20972 (Probe 1-AP)) and CD11b (Itgam) mRNA (VB6-15396 (Probe 6-AP)) (Affymetrix) were used for co-expression analysis which, with smaller modifications, was performed according to the QuantiGene ViewRNA ISH Tissue 2-Plex Assay Protocol by Affymetrix as described in Grebing et al. (2016) (link). After ISH the sections were counterstained with hematoxylin (Sigma-Aldrich), rinsed in tap water three times, dried for 1 h and coverslipped in Ultramount (Dako).