Imager d2 microscope

Panicles were fixed in FAA fixative (50% ethanol, 10% formaldehyde, 5% acetic acid) overnight at 4 °C. After dehydration under a gradient series of ethanol and xylene, panicles were embedded in paraplast plus (Leica, Wetzlar, Germany) and sectioned to a thickness of 7 µm using the RM2245 rotary microtome. The paraffin sections were dewaxed in xylene and rehydrated in an ethanol series. The TUNEL assay was performed using the DeadEnd Fluorometric TUNEL system (Promega, Madison, MI, USA, #G3250), according to the manufacturer’s instructions. Signals were observed and imaged using an Imager D2 microscope (Carl Zeiss, Oberkochen, Germany).

Samples of 50 mL were fixed with buffered, sterile-filtered paraformaldehyde (Penta, Prague, Czechia) to a final concentration of 1%, and 0.5 mL was filtered onto white polycarbonate filters (pore size 0.2 µm, Nucleopore, Whatman, Maidstone, UK). Cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) at concentration of 1 mg L⁻¹ [25 ]. Total and AAP bacterial abundances were determined using an epifluorescence Zeiss Axio Imager.D2 microscope equipped with Collibri LED module illumination system (Carl Zeiss, Jena, Germany). Ten microphotographs were taken for every sample under 325–370 nm excitation and 420–470 nm emission wavelengths for DAPI fluorescence (total bacteria), 450–490 nm excitation and 600–660 nm emission wavelengths for autofluorescence from Chl-a (algae and cyanobacteria), and combined 325–370 nm, 450–490 nm, 545–565 nm and 615–635 nm excitation and 645–850 emission wavelengths for autofluorescence from BChl-a (AAP bacteria). As some part of Chl-a autofluorescence is also visible in the infrared spectrum, only the IR-positive cells that did not show any autofluorescence from Chl-a were counted as AAP bacteria [26 (link)].