Vectashield anti fade medium with dapi

PND2 and 4 month old C57BL/6J mice were injected subcutaneously with 10 µg/g body weight EdU (Invitrogen) dissolved in 1xPBS. 24 h later, mice were sacrificed and hearts were collected and prepared according to above methods. Fixed tissue sections were blocked for 1 h at room temperature (1% BSA, 0.1% Cold water fish skin gelatin, 0.1% Tween 20 in PBS with 0.05% NaN₃), followed by use of Click-it EdU Kit (Invitrogen) to detect presence of EdU according to the manufacturer’s instructions. Sections were then mounted in Vectashield anti-fade medium with DAPI (Vector Laboratories) to detect cell nuclei. The total number of cell nuclei in one leaflet were counted using ImageJ cell counter. The number of EdU + cells were then counted and calculated as a percentage of total cells. An average of 9 leaflets were counted and averaged for each mouse, with a total n = 3. Statistical analysis was performed in GraphPad Prism 7.0a.

Cells in microscopic chambers were fixed with buffered 4% formaldehyde for 15 min and washed in PBS three times. Subsequently, cells were permeabilized by 0.3% Triton X-100 in PBS for 15 min and blocked by 5% BSA in PBS for 30 min. Samples were incubated with primary antibody for 60 min at ambient temperature, then 60 min with secondary antibody at ambient temperature. Finally, 25 µL of Vectashield^® antifade medium with DAPI (Vector Laboratories, Inc., 30 Ingold Road, Burlingame, CA 94010, USA) was added to each cell chamber for nuclear staining.

The FFPE sections (3 μm) were deparaffinized for fluorescence immunohistochemistry. For heat-induced antigen retrieval, tissue sections were immersed in 0.01 mol/L citrate buffer and treated in a microwave oven at 620 W. The sections were then blocked with 1% bovine serum albumin for 1 h at room temperature. Sections were then incubated with the primary antibody, anti-FOXP3 (1:50) or anti-CXCR4 (1:50), overnight at 4°C, washed with PBS, and incubated with donkey anti-rabbit-Alexa Fluor 488 (1:1000; Thermo Fisher Scientific, Tokyo, Japan), goat anti-mouse-Alexa Fluor 594 (1:1000, #ab150073, Abcam) for 30 min at room temperature and placed on slides using mounting medium with DAPI (Vectashield antifade medium with DAPI: Vector Laboratories).

Murine hair follicles for IF assays were obtained from HsdWin:NMRI mice (Envigo, UK). All samples were fixed in 10% neutral buffered formalin (Sigma-Aldrich) for 20 min and permeabilized with 0.5%TritonX-100 (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) in phosphate-buffered saline (PBS) for 10 min. After every step, samples were washed in PBS (2 x 2 min). Then they were blocked in PBS containing 1% bovine serum albumin (Sigma-Aldrich) for 20 min. The blocking solution was also used to dilute the antibodies. Samples were incubated in primary antibody solution overnight at 4° C. After washing with PBS, samples were incubated in secondary antibody solution for one hour. The primary and secondary antibodies are listed in Supplementary Tables 2, 3. Samples were washed in PBS and mounted using Vectashield antifade medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Images of mouse hair follicles were obtained using an Axio Observer.Z1/Cell Observer Spinning Disc microscopic system (Zeiss, Oberkochen, Germany) with a 63x oil objective. All images were processed using ZEN Blue Image processing software (Zeiss).

Metaphase preparations were fixed to slides and dehydrated through an ethanol series (2 min each in 2× SSC, 70%, 85%, and 100% ethanol at room temperature). Probes were diluted in a formamide buffer (Cytocell) with chicken hybloc (Insight Biotech) and applied to the metaphase preparations on a 37°C hotplate before sealing with rubber cement. Probe and target DNA were simultaneously denatured on a 75°C hotplate prior to hybridization in a humidified chamber for 72 h at 37°C. Slides were washed post-hybridization for 30 sec in 2× SSC/0.05% Tween 20 at room temperature and then counterstained using VECTASHIELD anti-fade medium with DAPI (Vector Labs). Images were captured using an Olympus BX61 epifluorescence microscope with cooled CCD camera and SmartCapture (Digital Scientific UK) system. In selected experiments, we used multiple hybridization strategies, making use of the Cytocell octochrome (eight-chamber) and multiprobe (24-chamber) devices. Briefly, labeled probes were air dried on to the device. Probes were rehybridized in standard buffer and applied to the glass slide (which was subdivided to correspond to the hybridization chambers), and FISH continued as above.

For histology experiments, unless otherwise stated, hearts were collected from mice at relevant timepoints, fixed in 4% paraformaldehyde, processed for paraffin embedding, and sectioned at 7 μm. For all antibodies (see Table 1), paraffin was removed through a series of xylene treatments, and tissue sections were rehydrated through a graded ethanol series and rinsed in 1x PBS. Sections containing the AoV were then subjected to antigen retrieval by boiling for 10 min in unmasking solution (Vector Laboratories, Newark, CA, USA #H-3300). Following this, sections were treated for 1 h at room temperature in blocking solution (1% BSA, 1% cold water fish skin gelatin, 0.1% Tween-20/PBS) and incubated overnight at 4 °C or 1 h at room temperature with indicated primary antibodies diluted in blocking solution or a 1:1 solution of 1x PBS and blocking solution.
For primary antibody detection, sections were incubated for 1 h at room temperature with appropriate secondary antibodies including Alexa Fluor 488 or Alexa Fluor 568 donkey α-rabbit, donkey α-goat, and donkey α-mouse diluted in 1x PBS (1:400, LifeTechnologies, Carlsbad, CA, USA), then mounted in Vectashield anti-fade medium with DAPI (Vector Laboratories, Newark, CA, USA #H-1500) to detect cell nuclei.