Antigenfix

Experiments were performed using the RNAScope Multiplex Fluorescent V2 Assay kit (ACDBio 323100). Probes targeting intronic regions for Hs-Cd5l (ACDBio 850511), Mfa-Cd5l (ACDBio 873211), Mm-Cd5l (ACDBio 573271), Mm-Flt3 (ACDBio 487861), Mm-Xcr1 (ACDBio 562371), Mm-Mafb (ACDBio 438531) and Mm-Mgl2-O1 (ACDBio 822901) were custom-designed and synthesized. They were then labelled with TSA opal 520 (PerkinElmer FP1487001KT), TSA opal 540 (PerkinElmer FP1494001KT), TSA opal 570 (PerkinElmer FP1488001KT), TSA opal 620 (PerkinElmer FP1495001KT) or TSA opal 650 (PerkinElmer FP1496001KT). Tissues were fixed for 16 hours in AntigenFix (Diapath P0016), dehydrated and embedded in OCT as described above. Slices were pre-treated with hydrogen peroxide for 10 min and protease III for 20 min. The recommended Antigen retrieval step was not performed in order to preserve epitope integrity. Probes were hybridized and amplified according to the manufacturer’s instructions. Slides were then stained for protein markers as described above.

Confocal staining was performed as described previously (Bonnardel et al., 2019 (link)). Immediately after sacrificing mice with CO₂, inferior vena cava were cannulated and livers were perfused (4 mL/min) with Antigenfix (Diapath) for 5 min at room temperature. After excision, 2-3 mm slices of livers were fixed further by immersion in Antigenfix for 1h at 4°C, washed in PBS, infused overnight in 34% sucrose and frozen in Tissue-Tek OCT compound (Sakura Finetek). 20 μm-thick slices cut on a cryostat (Microm HM 560, Thermo Scientific) were rehydrated in PBS for 5 min, permeabilized with 0,5% saponin and non-specific binding sites were blocked for 30 min with 2% bovine serum albumin, 1% fetal calf serum and 1% donkey or goat serum for 30 minutes. Tissue sections were labeled overnight at 4°C with primary antibodies followed by incubation for 1h at room temperature with secondary antibodies. When two rat antibodies were used on the same section, the directly conjugated rat antibody was incubated for 1h after staining with the unconjugated and anti-rat secondary and after an additional blocking step with 1% rat serum for 30 minutes. Slides were mounted in ProLong Diamond, imaged with a Zeiss LSM780 confocal microscope (Carl Zeiss, Oberkochen, Germany) with spectral detector and using spectral unmixing and analyzed using ImageJ and QuPath software.

Mouse heart tissue samples were fixed in Antigenfix (Diapath, Martinengo, Italy) at 4°C for 1 hour, then rinsed twice in PBS, immersed in 34% sucrose overnight, and embedded in Tissue-Tek OCT compound (Sakura Finetek) before being frozen. Sections of 20 µm thickness were sliced using a cryostat (Leica 1950), rehydrated in PBS for 5 minutes, permeabilized with 0.5% saponin, and blocked for 30 minutes with a mixture of 2% BSA, 1% FBS, and 1% donkey or goat serum. The heart sections were incubated with primary antibodies followed by suitable secondary antibodies. After mounting the slides in ProLong Diamond, imaging was performed using a Zeiss LSM780 confocal microscope (Carl Zeiss, Oberkochen, Germany) equipped with a spectral detector for spectral unmixing. Image analysis was carried out using Imaris software (Oxford Instruments, Zürich, Switzerland).

BM cells (50 to 100 μl resuspended in DMEM at a concentration of 10⁶ cells/ml) were loaded on alcian blue-coated coverslips (Sigma) and incubated for 20 min at 37°C to allow cell attachment. Twenty minutes Antigenfix (Diapath) was used for fixation. Once fixed onto coverslips, cells were washed with PBS and slides were mounted using ProLong Gold Antifade reagent containing DAPI (Thermo Fisher Scientific). Slides were observed with confocal microscope (Leica TCS SP8) as described before [18 (link)]. Image analyses were performed using the ZEN 2011 software.