*2.5. Immunofluorescence*

Sections of rat femur were fixed in paraformaldehyde (PFA (P6148, Sigma, Lezennes, France); 4% ( *w*/*v*) in PBS (Phosphate-Buffered Salin without Ca and Mg, GAUPBS0001, Eurobio, Les Ulis, France)). After three washes in PBS, the sections were permeabilized for 15 min with PBS containing 0.5% Triton X100 (*v*/*v*). The non-specific binding sites were blocked with Emerald Antibody Diluent (Sigma 936B-08) for 1 h. The sections were incubated overnight at 4 ◦C with the primary rabbit anti-CD68 (ab125212, Abcam, Amsterdam, The Netherlands) antibody at 1:1000 dilutions. Then they were washed in PBS and incubated with the secondary anti-rabbit Alexa Fluor 488 (A-21206, Thermo Scientific, Villebon sur Yvette, France) antibody at 1:500 dilution for 2 h at room temperature. Finally, sections were washed in PBS for 20 min and mounted using a Fluoroshield mounting medium with DAPI (Abcam, ab104139). Fluorescence was detected using an epifluorescence microscope DM6000 (Leica, Germany) equipped with monochrome and color digital cameras.

### *2.6. In Situ-DIG Hybridization*

For in situ-DIG hybridization, a couple of primers were chosen to ge<sup>t</sup> an approximately 1000 bp (β-actin (944 bp, CD68 1059 bp) PCR amplicon (Table S1A). β-actin and CD68 DNA primers were designed using ApE software (Table S1B). Two other primers, including 62 bp of the PCR product, were designed to recognize the T3 and T7 promoter sequences to perform the in vitro transcription (Figure 2). DNA oligos were synthesized by Eurogentec and were dissolved in ddH2O and stored at −20 ◦C.

**Figure 2.** Schematic representation of the PCR product (together with primer localization and orientation) used for in vitro transcription.

### 2.6.1. cRNA Probes for In Situ-DIG Hybridization

Frozen femur samples were homogenized in liquid nitrogen. RNA was isolated using RNeasy Fibrous Tissue mini kit (HB-0485, Qiagen, Courtaboeuf, France) according to the manufacturer's recommendations. RNA extracts were eluted with 20 μL RNase-free water. Reverse transcription (RT) was performed with oligo(dT) primers following the instructions of the Sensiscript transcription kit (205211, Qiagen). Reactions were carried out using 50 ng of RNA with 10 μM oligo(dT) primers, RNase inhibitor (2 IU) and Sensiscript reverse transcriptase. cDNA was synthesized at 37 ◦C during 60 min. This cDNA library was used for the amplification of the β-actin templates for in vitro transcription. These templates contained the T3 and T7 promoters. The cRNA labeling was generated by in vitro transcription with T3 and T7 RNA polymerases, both in antisense and sense direction. Sense probes were used as controls. The PCR fragments were purified by agarose gel electrophoresis and specific bands were isolated using PCR Clean-Up kit (740,609.10, Macherey-Nagel, Hoerdt, France).

Next, RNA was labeled using an in vitro transcription Kit (P1450, Promega, Charbonnières les Bains, France) according to manufacturer's recommendations. RNA was labeled with digoxigenin, by addition of a modified nucleotide, DIG-11-UTP. Transcription was carried out in buffer containing dNTPs, DIG-11-UTP, RNase inhibitor and RNA polymerase at 37 ◦C for 1 h. DNAse was then added and the mix further incubated for 30 min at 37 ◦C to degrade DNA.

A total of 10 μL (10 mg mL−1) transfer RNAs (1010945001, Roche, Boulogne-Billancourt, France) were added to the mix and the probes were precipitated with 10 M ammonium acetate (A1542, Sigma) and 100% cold ethanol overnight at −20 ◦C. After centrifugation at 15,000× *g* at 4 ◦C for 30 min, the pellet was rinsed with 70% cold ethanol. To improve probe penetration for probes longer than 500 nucleotides, hydrolysis of the probe is needed. For hydrolysis, the probe was suspended in 50 μL carbonate buffer (120 mM Na2CO3, 80 mM NaHCO3, pH 10.2) and incubated at 60 ◦C for 55 min. The reaction was stopped with a buffer containing 10 μL of 10% acetic acid, 12 μL 3 M sodium acetate (pH 4.8) and 312 μL 100% cold ethanol and incubated at −20 ◦C for at least 30 min. After centrifugation, the pellet was washed with 70% cold ethanol and suspended in 50% RNase-free formamide.

The effectiveness of labeling was analyzed by dot-blots. cRNA were dot-blotted on a nitrocellulose membrane (88018, Thermo Fisher Sci., Illkirch, France), and detected with an anti-digoxigenin (11093274910, Roche) DIG-specific antibody. From each serial dilution of the probes, 1 μL was spotted on a nitrocellulose membrane, dried and UV-crosslinked for 1 min. To prevent nonspecific antibody binding, the membrane was blocked with 1% bovine serum albumin (BSA; GAUBSA01, Eurobio, Les Ulis, France) in 100 mM Tris pH 7.5 and 150 mM NaCl (Tris-NaCl) buffer for 15 min. Afterwards the membrane was incubated for 30 min with anti-DIG antibody at 1:2000 dilutions in BSA/Tris-NaCl and then washed three times for 5 min in BSA/Tris-NaCl and once with Tris-NaCl. Finally, the membrane was stained with an NBT kit (NBT/BCIP; S3771, Roche) according to the manufacturer's recommendations.

### 2.6.2. Fixation and Pretreatment of Sections for In Situ-DIG Hybridization

All the following steps were performed under a laminar flow cabinet and under RNase-free conditions. The tissues were fixed in 4% ( *w*/*v*) in PBS/paraformaldehyde (PBS, *w*/*o* Ca and Mg, GAUPBS0001, Eurobio; PFA, P6148, Sigma) for 30 min, and then treated with 100% methanol for 15 min and air-dried. Sections were incubated in 0.125 mg mL−<sup>1</sup> Proteinase K (P2308, Sigma) in 200 mL 100 mM Tris pH 7.5 and 50 mM EDTA buffer for 10 min at 37 ◦C to degrade the proteins and to improve the probes' access to the target mRNA. Proteinase K reaction was stopped with 0.2% glycine (G7126, Sigma) in 1X PBS. Then sections were treated with 0.5% acetic anhydride (A6404, Sigma) in triethanolamine solution (0.1 M pH 8) to avoid non-specific hybridization. The sections were then washed twice for 2 min with PBS and dehydrated with successive baths of saline solution and ethanol: 30 s in 30% ethanol, 0.85% NaCl buffer; 30 s in 50% ethanol, 0.85% NaCl buffer; 30 s in 75% ethanol, 0.85% NaCl buffer; 30 s in 85% ethanol, 0.42% NaCl buffer; 30 s in 96% ethanol; 30 s in 96% ethanol; 1 min in 100% ethanol. Slides were then stored at −20 ◦C.

### 2.6.3. Prehybridization and Hybridization for In Situ-DIG

The sections were pre-hybridized for 2 h at 45 ◦C in a pre-hybridization buffer (50% formamide (GHYFOR0402, Eurobio), 0.5× sodium chloride citrate (SSC) (GHYSSC007, Eurobio) buffer, 50 μg mL−<sup>1</sup> heparin (H3393, Sigma), 100 μg mL−<sup>1</sup> transfer RNA and 0.1% (*v*/*v*) Tween 20 (822184, Merck). Finally, the sections were incubated overnight at 45 ◦C with the RNA probes (2 μL probe in 200 μL hybridization buffer (50% formamide, 100 μg mL−<sup>1</sup> transfer RNA, 7.5% (*v*/*v*) Tween 20, 8.5% NaCl, 20% dextran sulfate (Eurobio GHYDEX000T) and 2.5× Denhardt's Solution (50× stock, D2532, Sigma)), which were previously denaturized for 2 min at 80 ◦C in the hybridization buffer.

Non-specific hybrids were dissociated with following washes: 30 min in 0.1× SSC + 0.5% SDS at 45 ◦C, 2 h in 2× SSC + 50% formamide at 45 ◦C, 5 min in NTE (0.5 M NaCl, 10 mM Tris pH 8, 1 mM EDTA) at 45 ◦C, 30 min in NTE + 10 mg ml-1 Rnase A (10109169001, Roche) at 37 ◦C, 1 h in 2× SSC + 50% formamide at 45 ◦C, 2 min in 0.1× SSC at 45 ◦C and finally 15 min in PBS at RT.

### 2.6.4. Detection for In Situ-DIG

Immunodetection of the DIG-labeled probes was performed using an anti-DIG antibody coupled to alkaline phosphatase, as described by the manufacturer (11093274910, Roche). For the immunological detection step, the sections were incubated in a first buffer (0.5% Blocking reagen<sup>t</sup> (1110961176001, Roche) in 100 mM Tris pH 7.5 and 150 mM NaCl) for 1 h and in a second one (1% BSA in 0.5% (*v*/*v*) Triton X100, 100 mM Tris pH 7.5 and 150 mM NaCl) for 1 h to block unspecific sites. Sections were incubated with anti-digoxygenin antibody 1:1250 for 1 h, and then washed three times for 20 min with 1% BSA in 0.5% (*v*/*v*) Triton X100, 100 mM Tris pH 7.5 and 150 mM NaCl solution and next incubated for 15 min in the same solution without BSA. Finally, this solution was replaced with the last buffer (100 mM Tris pH 9.5, 100 mM NaCl and 50 mM MgCl2) for 15 min.

Staining was initiated at alkali pH. The sections were incubated for 1–2 days in a buffer containing 337 μL BCIP (5-bromo-4-chloro-3-indolyl-phosphate) and 225 μL NBT (Nitroblue tetrazolium chloride) in 50 mL solution (100 mM Tris pH 9.5, 100 mM NaCl and 50 mM MgCl2) until a blue precipitate adhering to the sections was formed. The reaction was stopped by adding a stop solution (10 mM Tris pH 7.5 and 5 mM EDTA) for 10 min. The DIG sections were observed with an epifluorescence microscope DM6000 (Leica, Germany) equipped with monochrome and color digital cameras while the HCR sections were observed with a confocal microscope (LSM700, Zeiss, Dresden, Germany).

### *2.7. In Situ-HCR Hybridization*

The HCR protocol of Choi and colleagues (2014, 2016) was performed with some modifications as described below to enhance mRNA localization in the femur of the rat [21,26].

### 2.7.1. β-Actin Probe Design for In Situ-HCR Hybridization

The probes were designed using ApE software. For each gene, five probes were designed. The entire gene sequence was used to localize the introns, and probes were designed exactly at the boundaries between two exons. This approach increased the capacity of probes to hybridize with the mRNA and not with the genomic DNA. A specific additional sequence was included to interact with the hairpin coupled with the fluorophore [21]. DNA oligos were synthesized by Eurogentec. Details of probe sequences are described in Table S1B. All oligos were dissolved in ddH2O and stored at −20 ◦C.

### 2.7.2. Fixation and Pretreatment of Sections for In Situ-HCR Hybridization

This process was common for both DIG and HCR in situ hybridization methods.

### 2.7.3. Prehybridization and Hybridization for In Situ-HCR

The sections were pre-hybridized for 10 min at RT in a hybridization buffer (50% formamide, 5× SSC, 9 mM citric acid pH 6, 50 μg mL−<sup>1</sup> heparin, 1× Denhardt's Solution, 0.1% (*v*/*v*) Tween 20 and 10% dextran-sulfate). Previously, the hybridization probes (2 pmol per slide) were denatured for 2 min at 80 ◦C. Finally, the sections were incubated in a hybridization buffer together with probes overnight at 45 ◦C. Nonspecific hybrids were dissociated with the following washes: 30 min in 0.1× SSC + 0.5% SDS at 45 ◦C, followed by 2 h in 2× SSC + 50% formamide at 45 ◦C, and then 2 min in 0.1× SSC at 45 ◦C and finally 15 min in PBS at RT.

### 2.7.4. Detection for In Situ-HCR

Sections were first incubated for 2 h at RT with an amplification buffer (5× SSC, 0.1% (*v*/*v*) Tween 20, 10% dextran-sulfate and 100 μg mL−<sup>1</sup> salmon sperm ADN) and subsequently for 12 to 16 h with the DNA hairpins marked with a fluorophore (Alexa Fluor488) (diluted in amplification buffer, as described previously). The hairpins were previously heated at 95 ◦C for 90 s and cooled to RT for 30 min. The sections were then

washed 2× 30 min in 5× SSCT (5× SSC and 1% (*v*/*v*) Tween 20) and 5 min with 5× SSC without Tween at RT.
