Roticlear

Slides were deparaffinized using Roticlear (A538.1 Roth) before rehydration in an ethanol series. Slides were then stained for 10 min in Weigert’s Haemotoxylin (Sigma HT1079) and resolved for 5 min in running tap water. After a wash in distilled water, slides were stained for 1 h in a 0.1% solution of sirius red (Sigma, 365548) in picric acid (Sigma, P6744-1GA) at room temperature. Slides were then dipped in 1% acetic acid solution twice and then dehydrated rapidly in an ethanol series. Slides were then incubated in Roticlear (Roth A538.1) before mounting with VectaMount (Vector Laboratories H-5000).

The leaflets were fixed in 4% Formalin (Huberlab, Aesch, Switzerland) for four hours before being embedded in paraffin (Merck, Darmstadt, Germany). For the overall tissue analysis, 5 µm sections of the samples were stained with hematoxylin and eosin for a general overview, as well as Movat’s pentachrome and Elastica van Gieson staining to visualize the multiple layers of heart valve leaflets. In addition, a CD90 antibody staining was performed to confirm recolonization of the decellularized tissue.
Before staining, the sections underwent a deparaffination and rehydration process starting with Roticlear (Roth, Germany), followed by a descending ethanol (Huberlab, Switzerland) series, and a final step in ddH₂O. After staining, the same steps were taken in reverse to dehydrate the samples. All the sections were mounted in Eukitt (Merck, Rahway, NJ, USA).

Cryosections were dried for 15 min at room temperature, before the embedding medium was removed with distilled water for 2 min. Hematoxylin and eosin solutions (#T865.2 and #X883.2 Roth, Germany) were used for the staining. In short, sections were incubated for 10 min in hematoxylin solution, washed for 15 min with warm and running tap water, followed by 2 min wash with aqua dest, and 1 min staining in eosin solution. Finally, we washed with running tap water, incubated the sections shortly in 70, 95 and 100% ethanol, twice for 2 min in Roticlear (#VA538.1; Roth, Germany), and mounted all sections with Cytoseal (#8312‑4, ThermoFisher, Germany).

Paraffin-embedded mice liver sections were deparrafinized by placing them consecutively in Roticlear (Carl Roth, Karlsruhe, Germany), 100% ethanol, and 95% ethanol. Sectioned tissues were boiled in 10 mM Na citrate buffer (pH 6.5) for antigen retrieval and blocked in 1% BSA supplemented PBS for 30 min. For the detection of CD115, Ly-6C, and Ly-6G in tissues from 6 h post-infected mice, samples were treated with Alexa Fluor 488 anti-mouse CD115 (CSF-1R) Antibody (1:100), Alexa Fluor 700 anti-mouse Ly-6C Antibody (1:100), and Brilliant Violet 421 anti-mouse Ly-6G Antibody (1:100), respectively (all three antibodies are from Biolegend, London, UK). About 0.1% saponin supplemented PBS was used to dilute antibodies, and SYTOX orange was used to detect DNA in the tissue samples. Alexa Fluor 350 labeled anti-mouse FH antibody (1:1,000) (Bioss antibodies, MA, USA) diluted in 0.1% saponin supplemented PBS was used for detection of FH deposition in 24 h post-infected tissues and SYTOX green (5 µM) was used to detect DNA in the probes. Images were captured using LSM 710 with ZEN 2011 (401/421 nm for Alexa Fluor 350 and Brilliant Violet 421, 488/525 nm for Alexa Fluor 488, 504/523 nm for SYTOX green, 547/570 nm for SYTOX orange, and 679/702 nm for Alexa Fluor 700). The experiment was repeated three times.

Cultured slices were fixed in 2 mL 4% paraformaldehyde (PFA; MERCK KGaA, Darmstadt, Germany) overnight at +4 °C. Slices were processed without decalcification using a Sakura Tissue Processor VIP 5E-F2 processor (Sakura, Tokyo, Japan) and subsequently embedded in paraffin. Five-micrometer serial sections were obtained using a Microtome HM 360 rotation microtome (Hyland Scientific GmbH, Berlin, Germany). Sections were incubated overnight at +60 °C, deparaffinized by two 10 min washes in Roticlear (CarlRoth, Karlsruhe, Germany), and then rehydrated in a series of descending alcohol concentrations (100% for 5 min and 95% and 70% for 2 min each) and a brief wash in distilled water. After staining, whole object slides were scanned with Aperio ScanScope (Leicabiosystems, Vienna, Austria). Afterward, images were generated with Image Scope software (v12.4.0.50.43, Leicabiosystems). Quantitative analyses were performed with ImageJ (v1.53f51) (U. S. National Institutes of Health, Bethesda, MD, USA). To assess the morphological differences/changes between the injured bone and their respective controls, the postnatal femur was empirically divided into distinctive zones—transition zone (TZ), middle zone (MZ), and deep zone (DZ)—based on the maturation and differentiation stage of chondrocytes and their cell arrangement [13 (link)].