Tcs spe inverted confocal microscope

Hemolymphs of 50 infected D. citri insects were collected and then were placed on Polysine adhesion slides (Thermo Fisher Scientific) and dried at room temperature. The samples were fixed in 4% paraformaldehyde for 2 h and treated with 0.2% Triton X-100 for 1 h as previously described (12 (link)) and then were immunolabeled with P8-FITC or actin dye phalloidin-Alexa Fluor 647 carboxylic acid (Thermo Fisher Scientific). The treated samples were examined with a Leica TCS SPE inverted confocal microscope.
Organs of D. citri insects were successively dissected, fixed, and treated with Triton X-100, as mentioned above. The samples then were immunolabeled with P10-FITC and finally detected using immunofluorescence microscopy.

For whole-mount immunostaining, ZF embryos taken at appropriate ages were fixed in 4% PFA for 1 h at room temperature then washed (4 × 5 min) in PBST with 1% triton X-100 (PSBT). While still in PBST, embryos were carefully dechorionated, then permeabilized in ice-cold acetone for 8 min. Following washes (4 × 5 min in PBST), the embryos were incubated in blocking buffer (PBS-T+10% FCS) for 1 h (RT) after which the buffer was changed and then repeated for another 1 h. Embryos were then incubated for 3 days at 4 °C in primary antibodies diluted in blocking buffer (1:200 and 1:500 for Brn-3a and Brn-3b rabbit polyclonal Ab; 1:500 of tropomyosin mAb and 1:200 of α-actinin mAb) with gentle rotation. Embryos were washed (3 × 1 h) in blocking buffer followed by 3 × 10 min in PBS. Fluorescent-tagged secondary antibodies (AlexaFluor 555 for tropomyosin or α-actinin mAb or AlexaFluor 488 for Brn-3a or Brn-3b rabbit pAb) were diluted 1:2000 in blocking buffer and incubated with embryos for 3 days at 4 °C, under sealed darkened conditions with gentle rotation. Embryos were then washed (3 × 10 min in PBST), mounted in 1% low melting point agarose on glass bottom culture dishes (MatTek, Peterborough, UK) and stored at 4 °C in the dark until confocal imaging could be undertaken (Leica TCS SPE inverted confocal microscope (× 20 magnification)).

Cells were grown in 8-well Millicell ez slide culture plates (Millipore, USA) and treated for 24 h with HTO (150 μM) in serum-free medium containing 1% BSA (bovine serum albumin) prior to stimulation for 30 min with EGF (100 ng/ml). The cells were then washed and fixed with 4% paraformaldehyde for 20 min at RT, washed again with PBS and incubated overnight at 4°C with an anti-EGFR IgG. Finally, the cells were washed with PBS and incubated at room temperature with alexa^® 488 conjugated anti-mouse IgG (Invitrogen, Thermo fisher scientific Inc, Waltham, MA, USA) for 1 h. The nuclei were stained with Hoechst (Thermo-scientific, USA) and the cells were visualized on a Leica TCS SPE inverted confocal microscope (Barcelona, Spain).

For visualizing RdFV infection to different tissues of R. dorsalis, the alimentary canal, salivary gland, and male or female reproductive organs were dissected from 30 RdFV-free or -positive R. dorsalis leafhoppers. The samples were fixed in 4% (v/v) paraformaldehyde in PBS for 2 h, and then permeabilized in 0.2% (v/v) Triton-X for 1 h. The samples were then immunolabeled with RdFV CP-rhodamine (0.5 μg/μl) and the actin dye Alexa Fluor 647 Phalloidin (0.1 μg/μl). Immunostained tissues were visualized using a Leica TCS SPE inverted confocal microscope.
For visualizing the association of HongrES1 with RdFV or RGDV in the male reproductive system, the reproductive organs were dissected from 30 RdFV-free, RdFV-positive, or RdFV and RGDV co-positive males. The samples were fixed, permeabilized, immunolabeled with HongrES1-FITC, CP-FITC, CP-rhodamine, or P8-rhodamine (0.5 μg/μl), and then processed for immunofluorescence microscopy.
For visualizing virus or HongrES1 association with sperms, mature sperms were excised from the testes of 30 RdFV-free, RdFV-positive, or RdFV and RGDV co-positive males, and then smeared on poly-lysine-treated glass slides. The sperms were successively fixed, permeabilized, immunolabeled with CP-rhodamine, P8-rhodamine, P8-FITC, HongrES1-FITC (0.5 μg/μl), stained with DAPI (2.0 μg/ml), and then processed for immunofluorescence microscopy.