**1. Introduction**

Vertebrate bone is a dynamically changing tissue that constantly adapts throughout life. For successful bone healing, coordinated cross talk is needed between inflammatory and bone-forming cells [1–5]. Nowadays, the exact mechanisms of bone regeneration remains to be elucidated [6–9]. Bones and bone marrow contain different types of macrophages: (i) erythroid island macrophages; (ii) hematopoietic stem cell macrophages; and (iii) osteoclasts [10]. Macrophages play an important role both in osteoblast-mediated bone formation [9] and in osteoclast development [10]. In addition, the newly-discovered osteal macrophages, so called "osteomacs", have a fundamental role during bone regeneration [6,9,11,12]. The exact role of these cells is still under study. The cytokines and other soluble factors secreted by macrophages can induce the bone formation in vitro [2,5,9,13]. Cytokines are the critical actors in coordinating an efficient repair of damaged bone tissue [14]. Examination of cytokine expressions during bone regeneration is essential for establishing new diagnostic and therapeutic approaches for bone tissue repair. Protein expression analysis of bone cells mostly uses immunofluorescence technique methods to detect proteins in situ [15]. In spite of immunolabeling being very convenient and well reproducible, there are some disadvantages, such as non-specific labeling with certain antibodies [16,17]. Additional difficulty is that cytokines are usually secreted, so the identification of cells producing this peptide or protein is problematic [16].

**Citation:** Nikovics, K.; Castellarin, C.; Holy, X.; Durand, M.; Morin, H.; Bendahmane, A.; Favier, A.-L. In Situ Gene Expression in Native Cryofixed Bone Tissue. *Biomedicines* **2022**, *10*, 484. https://doi.org/10.3390/ biomedicines10020484

Academic Editor: Mike Barbeck

Received: 27 January 2022 Accepted: 15 February 2022 Published: 18 February 2022

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In contrast, in situ hybridization (ISH) is one of the most suitable methods to investigate localization of gene expression in situ and based on the detection of mRNA product of genes involved in protein translation [16,18–21]. The basis of this technique is that complementary RNA and DNA sequences form hybrids with one another by hydrogen bonding. ISH is a very powerful technique; however, the probe design is complex, and the different steps are fastidious, needing a high level of optimization. An additional problem is that the conservation of the RNA as mRNA is very sensitive to degradation and RNase enzymes can be found everywhere [20].

The digoxigenin technique (in situ-DIG) is the most commonly used non-radioactive IHS method [22,23]. It is highly sensitive, but unfortunately only allows the analysis of a single gene in a single sample. In the 1980s, the fluorescence in situ hybridization (FISH) technique was published [24]. This method has the advantage of allowing the simultaneous analysis of several genes in the same sample, but is only suitable for the analysis of highly expressed genes [25]. Recently, a new method has been published in which in situ hybridization is coupled with the detection by hybridization chain reaction (in situ-HCR) [21,26–28]. One of the main advantages of this technique is its high sensitivity that makes it suitable for testing low-expression genes. In addition, several genes can be analyzed simultaneously. However, in situ-HCR is less efficient than the in situ-DIG method [25].

We found that fixation in buffered formalin, decalcification in EDTA and embedding in paraffin is a good compromise, as it provides not only a good morphology and excellent conditions for immunohistochemistry but also allows DNA- and RNA-based molecular studies [15,29,30] (Table 1).


**Table 1.** Comparison of the different approaches.

Cryosectioning of hard tissue has been introduced several decades ago [31] and was optimized by the tape technique described by Kawamoto et al. [32–34]. The main advantage of this system is that there is no fixation and embedding before section preparation, so it is much faster than the conventional method and more useful for in situ hybridization (Table 1). As mRNA is very sensitive to degradation, the challenge resided in the ability to cut cryofixed bone tissue and to preserve mRNA for analysis. To overcome these limitations, cryosectioning of the bone of rat was combined with ISH on the entire femur together with the muscle.

### **2. Materials and Methods**

### *2.1. Rat Animal Model*

All experiments were approved by the IRBA Institutional Animal Care and Use Committee (protocol 65 DEF\_IGSSA\_SP). Interventions were carried out in an accredited animal facility. 8-week-old (200 g average weight) male Sprague Dawley rats (Charles River Laboratories, Freiburg, Germany) were housed individually in cages, in a temperatureand light-controlled environment, with food and water ad libitum. Before collecting the

femurs with muscles, animals were euthanized at 12 weeks old with an overdose of sodium pentobarbital (150 mg/kg) administrated intraperitoneally.

### *2.2. Slides Coating*

Slides were manually coated in two steps using coating and pretreatment solutions (Leica Microsystems, Richmond, IL, USA). Slides were first pretreated with A solution (5 mL of A buffer concentrate (39475270, Leica, Wetzlar, Germany), 1.25 mL of 0.1 M acetic acid and 25 mL of acetone in 500 mL distilled water) (Figure 1A). After 30 min in dark, the A solution was completed with acetone (q.s.p. 500 mL), filtered and stored until 6 months at 4 ◦C in a dark bottle. Slides were immersed 3 times in A solution and pulled out diagonally to avoid streaks. Slides were kept overnight at RT or warmed at 90 ◦C for 5 min before the coating process. A total of 15 μL of B solution (39475271, Leica) was coated on pretreated slides to obtain a thin and homogenous coating (Figure 1B,C). The surface of the coating was adapted to match with the cryosection surface (Figure 1D).

### *2.3. Embedding and Cryosectioning of Entire Femur of the Rat*

RNase-free instruments, materials and buffers were used to collect bone samples. After euthanasia of the rat, the whole femur was cleaned rapidly and a part of the muscles around the bone was kept. The femur was placed at the bottom of the embedding mold and covered with cryomoun<sup>t</sup> medium (CM) (00890-EX, HistoLab, Askim, Norvege). Samples were snap-frozen with 2-methylbutan cooled in liquid nitrogen to obtain a block (Figure 1E). When entirely frozen, the sample was transferred on dry ice to −80 ◦C, wrapped in foil aluminum. Then, it was stored at −80 ◦C until further processing. A cryostat (Cryostat FSE Shandon, Thermo Electron Corporation, San Diego, CA, USA) with a Leica CryoJane (9194701, Leica) system was used for cryosectioning. The block was fixed with cryomoun<sup>t</sup> medium (Figure S1A–D). Tools were precooled within the cryostat to avoid warming up the sample during block trimming (Figure 1F). The surface of the adhesive film (39475214, Leica) was adapted to the surface of the block (Figure 1G) and CryoJane tape transfer system was applied (39475205, Leica) to obtain high-quality sections.

The following points are crucial to the success of the experiment:

Briefly: (i) Positioning the adhesive film on the surface of the block (Figure S1E). (ii) Applying the roller while exerting a certain force to improve the adhesion of the film (Figure S1F). (iii) Cutting the sample slowly and uninterruptedly (Figure S1G). Obtaining 5 μm tissue sections on the adhesive film (Figures 1H and S1H). (iv) Transferring the section from the film to a standard histological slide, manually pretreated with Leica's A and B solutions (Figure S1I). (v) Fixation of the section on the coated side of the slide by CryoJane UV flash system (Figure S1J–L). (vi) Removal of the adhesive film (Figure S1M) and optimization the transfer of the cryo-section (Figure S2A–F). Four points are decisive to ensure good quality of the cryosections: (1) The CM block is very important for a good section (the tissue alone does not adhere well to the film). The film must be in contact with the CM and with the tissue. (2) The entire surface of the adhesive film must be in contact with the CM block. The film must not be wider than Sections 3 and 4. Very important is to hold the bottom of the film with a pair of pliers when cutting—this avoids heating the film which must remain cold and is safer (Figure 1M).

### *2.4. Histological Staining*

Hematoxylin and phloxin (HP) staining was performed as followed: the sections were incubated in several successive baths: 40 s in hemalum (11487, 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 phloxin (15926, Merck) 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, 1 min in 100% ethanol. At the end, nuclei were colored in blue and cytoplasm in pink (Figure 1I–K).

**Figure 1.** Cryo-fixation and cryo-section steps. Slides were manually coated with (**A**) A and (**B**) B buffer. (**C**) Thin B buffer layer on the slide surface. (**D**) Adjusted coating of the manually prepared slide compared with the manufactured one. (**E**) Entire cryo-embedded and trimmed femur of rat. (**F**) Cryo-bar to precool slides, adhesive film and tweezers. (**G**) Several surface size of adhesive film. (**H**) Thin (5 μm) cryo-section of femur deposed on a pre-coated slide. (**I**,**J**) Histological coloration of femur stained with Hemalin–phloxine–saffron. (**K**) Expanded view: high magnification image of the area within the red rectangle in image (**J**). (**L**) Von Kossa staining of the mouse and rat bone. (**M**) Upper panel: technical practices to avoid; down panel: to improve the result in comparison with the appropriate practices.

Von Kossa staining was performed as followed: Sections were rinsed with distilled water and incubated for 30 min in the dark with silver nitrate solution (1 g silver nitrate in 100 mL distilled water) at room temperature. After washes with distilled water, the sections were incubated under UV for one hour (sections should be covered with distilled water). Finally, the short passage in 95% and 100% ethanol, followed by xylene, were carried out. For the good conservation of the cryo-section, Eukitt mounting solution was used (Figure 1L).
