Goat anti rabbit secondary antibody conjugated to alexa fluor 488

Indirect immunofluorescence microscopy was performed according to standard procedures (Cai et al., 2011 (link)). Briefly, samples were fixed with 3% paraformaldehyde in PM buffer (50 mM PIPES, pH 6.9, 1 mM EGTA, 0.5 mM MgCl₂) for 30 min, washed with PM for 10 min and then incubated with 1.5% cellulysin (Sigma) for 7 min in the dark. After two washes in PM buffer, samples were incubated with the primary antibodies to callose synthase (Cai et al., 2011 (link)) at a dilution of 1:50. Antibodies were incubated at 4°C overnight. After two washes with PM buffer, samples were incubated in the dark with a goat anti-rabbit secondary antibody conjugated to Alexa Fluor 488 (Invitrogen) diluted 1:150 for 45 min. After two washes in PM buffer, samples were placed on slides and covered with a drop of Citifluor. Observations were made using a microscope Zeiss AxioImager equipped with structured illumination and a 63x objective; images were captured with an AxioCam MRm camera using the software AxioVision. In controls, primary antibodies were omitted.

IPEC-1 cells were seeded onto glass microscope coverslips (Corning, MA, USA). To assess the effect of inhibitors on H₂O₂-induced barrier function, cells were pretreated with SB202190, PD98059, or Bay11-7082 for 1 h and then challenged with H₂O₂ when cells reached 70%-80% confluency. All cells were fixed with 4% paraformaldehyde for 10 min at room temperature (RT) and permeabilized with 0.2% Triton X-100 in PBS at RT for 10 min. The cells were blocked with 1% bovine serum albumin in PBS for 30 min at RT and then incubated with anti-claudin-1 antibody overnight. After being washed five times in PBS, the cells were incubated for 2 h at RT with a goat anti-rabbit secondary antibody conjugated to Alexa Fluor 488 (Invitrogen, CA, USA), followed by counterstaining with 4,6-diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO, USA). The coverslips were mounted onto glass microscope slides using mounting buffer, and the stained cells were visualized using a fluorescent confocal laser scanning microscope (Carl Zeiss, Gottingen, Germany).

Measurement of α-SMA using immunofluorescence staining was performed as previously described [12 (link)]. Briefly, after being placed in 24 well plates, mCFs were washed with PBS twice and then were blocked with 1 % bovine serum albumin at room temperature for 30 min. Then the cells were incubated overnight with rabbit anti-α-SMA primary antibody (1:100, Abcam). After being washed with PBS, the cells were performed with goat anti-rabbit secondary antibody conjugated to Alexa Fluor 488 (1:400, Invitrogen) for 1 h under dark condition. Finally, the cells were counterstained with DAPI (Sigma) for 10 min, and were analyzed with an immunofluorescence microscope.

For analyses of the L2 dendritic tree perimeter, we used the progeny of crossing the 21D-Gal4 line with the UAS-mCD8-GFP line (Fig 1). To inhibit the expressions of the Tor, Atg5 or Atg7 genes in L2 monopolar cells, additional crosses of 21D-Gal4>UAS-mCD8-GFP line with UAS-Tor-RNAi, UAS-Atg5-RNAi or UAS-Atg7-RNAi transgenic lines were performed. Seven-day-old males from the above crosses were decapitated at four time points: ZT1, ZT4, ZT13 and ZT16. The heads were fixed in 4% paraformaldehyde in 0.1 M PBS and cryoprotected overnight in 25% sucrose solution. Cryostat sections (14 μm in thickness) were prepared. To enhance GFP fluorescence in L2 cells, frozen sections were immunostained with rabbit polyclonal anti-GFP primary serum (Nouvos Biological, diluted 1:1000) followed by goat anti-rabbit secondary antibody conjugated to AlexaFluor 488 (Invitrogen, diluted 1:1000). The cryosections were mounted in Vectashield medium (Vector). Sections of the distal lamina were examined using a Zeiss Meta 510 Laser Scanning Microscope. The images were deconvolved using Huygens Professional software. Changes in the perimeter of the L2 dendritic trees were examined by tracing the outline of the dendrites and the axons of L2 cell cross-sections. Measurements were performed using ImageJ (v. 1.4 g with Java 1.6.0_05) software.

Cells were seeded on glass coverslips (Corning, NY, USA) and firstly pretreated with 0 or 10 μmol/L Que for 24 h, and afterwards treated with or without 0.5 μg/mL DON for another 48 h. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. The cells were blocked with 1% bovine serum albumin in PBS for 30 min and then incubated with anti-claudin-1 (#519000, Invitrogen, Waltham, MA, USA) antibody and anti-occludin (#abs131224, Absin) antibody overnight. Cells were then incubated with a goat anti-rabbit secondary antibody conjugated to Alexa Fluor 488 for 2 h (Invitrogen), followed by counterstaining with 4,6-diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO, USA). The coverslips were mounted onto glass microscope slides using mounting buffer, and visualized using a fluorescent confocal laser scanning microscope (FV10i, Olympus). The fluorescence intensity was quantified by the Olympus Flouview ver 3.1b software.