In Situ Gene Expression in Native Cryofixed Bone Tissue

Abstract

Bone is a very complex tissue that is constantly changing throughout the lifespan. The precise mechanism of bone regeneration remains poorly understood. Large bone defects can be caused by gunshot injury, trauma, accidents, congenital anomalies and tissue resection due to cancer. Therefore, understanding bone homeostasis and regeneration has considerable clinical and scientific importance in the development of bone therapy. Macrophages are well known innate immune cells secreting different combinations of cytokines and their role in bone regeneration during bone healing is essential. Here, we present a method to identify mRNA transcripts in cryosections of non-decalcified rat bone using in situ hybridization and hybridization chain reaction to explore gene expression in situ for better understanding the gene expression of the bone tissues.

Macrophages Characterization in an Injured Bone Tissue

“…In our case, paraffin embedding was not possible because the muscle significantly slowed down (around 5 weeks) the decalcifying solution (ethylene diamine tetra acetic acid (EDTA)) from penetration and did not allow the use of ISH techniques in these sections (data not shown) due to the mRNA degradation. To avoid this problem, an improved version of the CryoJane tape transfer system was developed in our laboratory to obtain the cryosections of the entire femur surrounded by muscle, which was suitable for ISH techniques [ 49 ].…”

“…Unfortunately, the expression of cytokines is usually low. The in situ HCR [ 49 , 50 , 51 , 52 , 53 ] method is a promising new method to study gene expression in bone tissues. This technique allows the detection of multiple mRNAs also in the same section and is suitable for the visualization of mRNA with low expression.…”

“…Embedding and cryosectioning methods were performed as described in [ 49 ]. RNase-free instruments, materials, and buffers were used to collect bone samples.…”

“…Histological staining was performed as described in [ 49 ]. Hematoxylin and phloxin (HP) staining was performed as follows: the sections were incubated in several successive baths: 40 s in a hemalum (11,487, Merck, Darmstadt, Germany) buffer (0.2 g hemalum, 5 g aluminum potassium sulfate in 100 mL distilled water), 3 min in water, 30 s in a phloxin (15,926, Merck, Darmstadt, Germany) buffer (0.5 g phloxin in 100 mL distilled water), 1 min in water, 2 min in 70% ethanol, 30 s in 95% ethanol, 1 min in 100% ethanol, and 1 min in 100% ethanol.…”

“…In situ hybridization methods were performed as described in [ 49 ]. Oligos designed for in situ hybridization experiments are listed in Table S2 .…”