Fluorescent brightener 28 fb28

Sections were treated for 30 min at room temperature (RT) with a blocking reagent consisting of 2% (v/v) foetal calf serum and 2% (w/v) bovine serum albumin in PBS (pH 7.2). Next, sections were incubated at RT for at least 1.5 h with specific primary monoclonal antibodies (Table 1), diluted 1:20 in a blocking reagent, rinsed 3×10 min with the blocking reagent and incubated at RT for at least 1.5 h with AlexaFluor 488 goat anti-rat IgG (Jackson Immuno-Research Laboratories) diluted 1:100 in the blocking reagent and used as the secondary antibody. After several washes with the blocking reagent and PBS, the sections were stained with 0.01% (w/v) fluorescent brightener 28 (FB28) (Sigma-Aldrich) in PBS for 5 min, then with 2 μg/ml DAPI in PBS (5 min at RT) after which they were thoroughly rinsed with PBS and sterile distilled water. Dried slides were mounted in a Fluoromount (Sigma-Aldrich) anti-fading medium. Negative controls were performed for each antibody used by omitting the primary antibodies. All images were taken using a Zeiss Axio Imager Z2 microscope equipped with an AxioCam Mrm monochromatic camera (Zeiss) with the corresponding software and narrow band filters for AlexaFluor 488 and DAPI.

To study the impacts of LmFTZ-F1s RNAi on chitin formation, microsections and chitin staining of cuticle were conducted as previously reported [33 (link)]. Briefly, paraffin sections (5 μm) of the second abdominal cuticle from dsGFP- and dsLmFTZ-F1s-injected nymphs were prepared on day 5 of the third instar. Then, the chitin was stained with Fluorescent Brightener 28 (FB28) (Sigma, Inc. St Louis, MO, USA) (1 mg/mL), and the nuclei were labeled with SYTOX™ Green Nucleic Acid Stain (Thermo Fisher Scientific, Waltham, MA, USA) (25 μg/mL) [34 (link)]. An LSM 880 confocal laser scanning microscope (Zeiss, Inc., Oberkochen, Germany) was used to capture images of the stained samples.