Anti digoxigenin fitc antibody

Genomic DNA was extracted from 4 g of young leaves using the CTAB method of Rogers and Bendich [34 (link)]. To create a probe, the total genomic DNA of A. roylei was fragmented with Labsonic^® M (Sartorius, Göttingen, Germany) and labeled with Dig-11-dUTP (Roche Diagnostics Gmbh, Mannheim, Germany) according to the manufacturer’s instructions. The genomic DNA of A. cepa was used as a block DNA. DNA denaturation and in situ hybridization steps were performed according to Schwarzacher and Heslop–Harrison [35 ]. The hybridization mix contained 50% (v/v) deionized formamide, 10% (w/v) sodium dextran sulfate, 2xSSC, 0.25% (w/v) SDS, 1.0 ng/µL probe DNA and 30µg/µL block DNA. A 78% stringency washing was applied. The Dig-11-dUTP labeled DNA was detected with anti-Digoxigenin-FITC antibody raised in sheep (Roche Diagnostics Gmbh, Mannheim, Germany) and amplified with anti-sheep-FITC antibody raised in rabbits (Vector Laboratories, Burlingame, California, USA). A. cepa chromosomes was counter-stained with DAPI in Vectashield antifade mounting medium at a concentration of 1:20 (Vector Laboratories, California, USA).

The combination of immunofluorescence and FISH experiments was performed as previously described with slight modifications60 (link). Briefly, the paraffin embedded sections were deparaffinized and incubated with primary mouse anti-CD68 monoclonal antibody(sc-17832, Santa Cruz; 1:200). Alexa Fluor 555-conjugated donkey anti-mouse IgG (Invitrogen, CA, USA) was used. The sections were washed twice with PBS and dehydrated by ethanol. The sections were rehydrated in 50% formamide in 2 × SSC for 5 min and prehybridized in 2 × SSC for 1 h at 42 °C. Then, the sections were hybridized with 20 nM 5' digoxigenin-labelled LNA control probe and miR-142-5p or miR-130-3p probe (Exiqon) overnight at 42 °C, then rinsed twice with 5 × SSC at room temperature, washed three times in 2 × SSC/50% formamide at hybridization temperature for 20 min and washed four times in PBS with 0.1% Tween 20 (PBST). Sections were blocked for endogenous peroxidase activity (30 min in 3% H₂O₂ in PBST) and incubated with blocking buffer (0.5% blocking reagent with 10% serum) for 1 h and with anti-digoxigenin-FITC antibody (Roche, 1:10,000) overnight at 4 °C. Sections were washed twice in PBST for 5 min and counter stained with DAPI (Invitrogen). Cells stained with the indicated probe were calculated per field of view and at least 10 fields per section were evaluated at 400 × magnification.