Histochoice clearing agent

Formalin fixed and paraffin embedded (FFPE) breast tissue sections from breast reduction surgery of a healthy Danish woman was kindly provided by IN-Lab Medico Aps, Virum, DK. Deparaffinization was performed with HistoChoice Clearing Agent (Sigma-Aldrich, St Louis, MO, USA).

4 µm sections were serially dewaxed 3×10 min in Histochoice clearing agent (Sigma), 2×5 min in 100% ethanol, and 3 min in 90%, 70% and 50% ethanol. Sections were then immersed in water for at least 5 min and boiled in citrate buffer (pH 6.0) for 2 min. Next, sections were rinsed in PBS and blocked overnight with 30% FCS in PBS 0.1% Triton X-100. Sections were then incubated with the corresponding primary antibodies in PBS 10% FCS: Rabbit anti mouse CD31 (1∶100, Abcam), rabbit anti mouse VE-cadherin (1∶800, Abcam) or monoclonal Cy3-conjugated antimouse α-SMA (1∶1000, Sigma). CD31 was detected using the DAB method (Biogenex), whereas VE-cadherin was incubated in cy3-conjugated goat anti rabbit (1∶500, Life Technologies). For immunocytochemistry, MEECs and MEFs were cultured for two days in gelatin coated coverslips, fixed for 15 min in 4% PFA, permeabilized for 10 min with PBS 0.25% Triton X-100, blocked in PBS 10% FCS and 0.05% Tween 20 (PBST) for one hour at room temperature, incubated with PBST containing rabbit antimouse CD31 (1∶50, Abcam) overnight at 4°C, washed 3×10 min in PBST, and incubated with cy3-conjugated goat anti-rabbit (1∶500, Life Technologies) for two hours at room temperature. Cells were photographed upon mounting with Dapi containing medium (Life Technologies).

In total, 2.5 μm sections of TMAs and paraffinized cell pellets were placed on glass slides and dried for 10 min at 60 °C, dewaxed for 10 min in HistoChoice Clearing Agent (Sigma, H2779), and rehydrated by consecutive washes in ethanol: 100%, 95%, 70%, and 40% v/v for 30 sec each. Heat-induced antigen retrieval was performed at 95 °C for 40 min with Dako Target Retrieval Solution of pH 9 (Dako, S2367). After cooling at room temperature, the slides were incubated 2.5% BSA (Fluka Analytical, 05488) in TBST 0.1% for 1 hour at room temperature. Anti-ADPr [31 (link), 32 ] (rabbit, 1:500 dilution) and anti-ATP5a antibody (Abcam, mouse, 1:250) diluted in the blocking solution were incubated overnight at 4 °C. Secondary antibody Alexa Fluor 488 AffiniPure goat anti-mouse (Jackson ImmunoResearch, 115–545–003, 1:500 dilution) and Cy3 AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch, 111–165–144, 1:250 dilution) were incubated for 2 hours at room temperature. Nuclear counterstaining was performed DAPI (Biolegend, 422801, 1:10,000 dilution). Microscope cover glasses (Menzel–Glaser, 7001023) were mounted with Mowiol mounting medium.

The procedure was conducted essentially as described before [16 (link)]. Pieces of approximately 4 × 4 × 6 mm of whole rye, barley, wheat, oat, rapeseed, sunflower (containing seed coat, aleurone layer and endosperm), cassava and DORB were fixed in Karnovsky’s fixative and washed in 0.1 M cacodylate buffer pH 7.3 and in demineralized water. The samples were then dehydrated in a graded series of ethanol before infiltration into melted paraplast (paraffin) at 60°C using Histochoice clearing agent (Sigma-Aldrich). Paraffin-embedded samples were sectioned into 7–10 μm thick sections on a 2030 Biocut microtome (Reichert-Jung, AU). De-paraffination was performed with Histochoice and a graded series of ethanol. Samples were stained with Calcofluor White for β-glucan and cellulose visualisation, and with Pontamine Fast Scarlet 4B for cellulose and xyloglucan visualisation.

For the infection assays, the MDBK cells seeded in 24-well plates were incubated to 80% confluence. The cells were then infected with EV-F7 SWUN-AB001 at a multiplicity of infection (MOI) of 0.01, and re-incubated for 6, 12, 24 and 36 h. Mock infected cells were included as the controls.
As a measure of viral replication, an immunofluorescence assay was used to confirm the status of the viral infection at 6, 12, 24 and 36 h post-infection using a previously described method [11 (link)]. Briefly, the MDBK cells grown on Lab Tek chamber slides (Nunc) for 24 h were infected with SWUN-AB001 (MOI, 0.01) for 6, 12, 24 or 36 h, after which they were fixed with HistoChoice Clearing Agent (Sigma-Aldrich), permeabilized using 0.1% Triton X-100, and blocked with 1% bovine serum albumin. The cells were then incubated with an anti-viral antiserum prepared from EV-F7 strain SWUN-AB001 (1:100), followed by incubation with Alexa Fluor 488 anti-rabbit secondary antibody (1:1000) (Invitrogen). Nuclei acids were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen). The chamber slides were then mounted onto glass slides using Fluorescence Mounting Medium (Dako), and the cells were observed and imaged using a fluorescence microscope (OLYMPUS IX73). All experiments were performed at least three times.

Freshly harvested seeds of 4, 5, 7, 9, and 11 DAP were dehusked, fixed, and dried in ethanol series (as mentioned in the previous section). Ethanol was gradually replaced with xylene by keeping the tissues in increasing concentration of xylene and decreasing concentration of ethanol in the percentages of 25:75, 50:50, and 75:25. Next, two rounds of treatment with 100% xylene were given to the seeds at room temperature for 1 h on the rocker, followed by infiltration with paraplast X-TRA (Sigma-Aldrich; USA) by adding molten paraplast every 2–3 h at 60 °C for 3 days. The wax-infiltrated tissues were then embedded into molds (Yorko®; India), and 10-μm sections were cut for 4, 5, and 7 DAP and 15-μm sections were cut for 9 and 11 DAP using rotary microtome (Leica Biosystems, Germany). Paraplast was removed from the sections by immersing the slides in HistoChoice® clearing agent (Sigma-Aldrich; USA) for 1 h. Sections were stained with 0.01% toluidine blue-O for 10 min or 0.1% Coomassie brilliant blue/CBB [0.25% CBB in 50% methanol and 10% acetic acid] for 20 min or 2% I2/KI solution (2 g KI and 0.2 g Iodine in 100 ml MQ) for 2–5 min. The sections were mounted using D.P.X. mountant (Himedia®, India) and were visualized under light microscope (Eclipse 80i, Nikon; Japan) at × 20 magnification.