Microm hm 340e microtome

A 478‐bp fragment of GhNSP cDNA was amplified from the cDNA, which was made from the stage 8 anther of 1355B plants, with the primers GhNSP‐S and GhNSP‐AS (Table S1). The PCR product was cloned into the pGEM‐T‐Easy vector (Promega, Madison, WI, USA) and sequenced. Sense and antisense probes were transcribed in vitro from the T7 or SP6 promoter with respective RNA polymerases using the digoxigenin RNA‐labelling kit (Roche). Tissue sections were prepared as described by Min et al. (2013 (link)). In brief, samples were fixed in FAA [10% formalin, 5% acetic acid and 50% ethanol (v/v) in RNAase‐free water]. After dehydration and embedding of the tissue in paraffin wax, the sample blocks were sectioned into 10‐μm slices using the microm HM 340E microtome (Thermo Scientific, Waltham, MA, USA) and were applied to RNAase‐free glass slides. The sections were then dewaxed, rehydrated, prehybridized, hybridized and visualized as described by manufacturer’s instructions. Hybridization was detected by using the antidigoxigenin‐alkaline phosphatase conjugate (Roche, Mannheim, Germany), visualized by incubation with NBT/BCIP stock solution (Roche), and images were captured using a Zeiss Axio Scope‐A1 microscope.

Briefly, a 150–200 bp fragment was amplified from the H05 cDNA. The PCR product was cloned into the pGEM‐T‐Easy vector (Promega, USA) and sequenced. Sense and antisense probes were transcribed in vitro from the T7 or SP6 promoter with respective RNA polymerases using the digoxigenin RNA labelling kit (Roche, Germany). Tissue sections were fixed in 50% FAA [10% formalin, 5% acetic acid, and 50% ethanol (v/v) in RNase‐free water]. After dehydration and embedding of the tissue in paraffin wax, the sample blocks were sectioned into 8‐µm slices using the microm HM 340E microtome (Thermo Scientific, USA) and were applied to RNase‐free glass slides (Solarbio, China). Hybridization was detected by using the antidigoxigenin‐alkaline phosphatase conjugate (Roche, dilution 1:1000), visualized by incubation with NBT/BCIP stock solution (Roche, dilution 1:50), and images were captured using a Zeiss Axioscope A1 microscope (Carl Zeiss, Germany). Primers are listed in Table S8 (Supporting Information).