Cyanine 5

Wasf1 and WASF1 expression in adult male mice of all four genotypes was visualized by RNAscope, a chromogenic in situ hybridization technique, following the RNAscope Multiplex Fluorescent Reagent Kit v2 from ACD Bio (CA, USA). Fresh-frozen brains were sliced sagittally (lateral 1.2 mm Bregma) at a thickness of 20μm and mounted onto charged slides. Slices were then fixed by submersion into 4% PFA in 1X PBS and dehydrated by serial submersion in 50%, 70%, and 100% ethanol. Slices were then incubated in hydrogen peroxide before being treated with RNAscope Protease IV. The multiplex fluorescent assay was then performed: slices were hybridized to negative control (reference ID 320871, ACD Bio), positive control (ref ID 320881), or both human WASF1 (ref ID 533691) and mouse (ref ID 533701) Wasf1 probes; amplification reagents were then used to incubate the slides to increase signal strength; finally, fluorescent signals were developed using TSA Plus cyanine 3 (PerkinElmer, Llantrisant, UK) for channel 1 and cyanine 5 (also PerkinElmer) for channel 2 using channel-specific horseradish peroxidases. Vectashield with DAPI (Vector Laboratories, CA, USA) was then applied to the slices before glass coverslip placement. Slides were dried overnight and then imaged with an Axio Scan.Z1 slide scanner (Zeiss, Oberkochen, DEU) at 20X magnification.

Harvested tissues were fixed for 1 day in 4% paraformaldehyde and PBS directly after sacrifice. After fixation, the bones were rinsed and decalcified with 10% EDTA (pH 7.2–7.4) for 2 weeks on a shaker. The specimens were subsequently processed to a thickness of 5 µm. Immunohistochemistry (IHC) was performed using the MACH 4 Universal HRP-Polymer system (Biocare Medical, LLC, Pacheco, CA). The slides were then incubated with the following primary antibodies of interest: pERK1/2 (1:500), pCaMKII (1:200), pAKT1 (1:200), pPKA (1:200), pCREB (1:200), pNF-kB (1:200), pcMyc (1:200), and RANKL (1:200). Reactions were then visualized with diaminobenzidine (DAB). The specimen sections were counterstained with hematoxylin. All staining was performed as instructed by the manufacturer. ImageJ software (NIH) was used to quantify the number of positively stained cells. In addition, multiplex immunohistochemistry was performed using fluorescein, Cyanine 3, and Cyanine 5 dyes (PerkinElmer, Boston, MA) according to the manufacturer’s instructions. Histologic images were captured using a Cytation 5 Imaging Reader (Biotek, Winooski, VT), and osteoblasts highly expressing pERK1/2 were manually counted by researchers (JB and LL) under blinded conditions.

RNA-ISH was performed on fresh 3-μm formalin-fixed paraffin-embedded tissue sections using the RNAscope Multiplex Fluorescent Reagent Kit version 2 for target detection (#323100, Advanced Cell Diagnostics) according to the manual. Briefly, tissue sections were baked for 1 hour at 60°C, then deparaffinized, and treated with hydrogen peroxide for 10 min at room temperature. Target retrieval was performed for 15 min at 98°C, followed by protease plus treatment for 15 min at 40°C. All RNAscope probes (tables S3 and S4) were hybridized for 2 hours at 40°C, followed by signal amplification, and development of horseradish peroxidase channels was performed according to the manual. TSA Plus fluorophores fluorescein (1:750 dilution), Cyanine 3 (1:1500 dilution), and Cyanine 5 (1:3000 dilution) (NEL744001KT, PerkinElmer) were used for signal detection. The sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted with the ProLong Gold Antifade Mountant (P36930, Invitrogen). Images were generated using 3DHISTECH Pannoramic 250 Flash II digital slide scanner at the Genome Biology Unit supported by HiLIFE and the Faculty of Medicine, University of Helsinki, and Biocenter Finland. All samples were scanned using ×40 magnification with extended focus and seven focus levels.