Assessment of gene and protein expression in tissue sections is instrumental in medical research. However, this is often challenging to perform on skeletal tissues that require prolonged decalcification and have poor adhesion to slides. In this study, we optimized selected steps of in situ hybridization (ISH), immunohistochemistry (IHC), and immunofluorescence (IF) for formalin fixed and decalcified skeletal tissues. Sections from distal femur of 6-, 8- and 14- week-old rats injected with BrdU with or without a hemizygous eGFP transgene expressed under the control of a ubiquitous promotor were used. We report that proteinase K digestion is critical for the sensitivity of ISH, as concentrations that were too strong and too mild both resulted in loss of signal. In addition, intensified RNase A digestion removed nonspecific riboprobe-mRNA hybrids. Furthermore, enzymatic antigen retrieval using proteinase K provided more consistent results in IHC and can therefore be a useful alternative to heat induced epitope retrieval (HIER) for skeletal tissues where such treatment often damages the morphology. A mild proteinase K digestion also improved IF detection of GFP and worked well for double labeling IF of GFP and osteocalcin on frozen sections of formalin fixed and decalcified rat bones while maintaining morphology. In summary, this study provides strategies to improve protocols for enzymatic digestion in ISH, IHC, and IF for skeletal tissues and also demonstrates the importance of careful optimization and validation with the use of these techniques.