A12380

Immunofluorescence microscopic imaging was carried out using the following steps: the apical and basal channels of the chips were gently washed with PBS and fixed with 4% paraformaldehyde (Electron Microscopy Sciences, 157-4) in PBS for 30 min, then washed twice with PBS and kept at 4 °C. The fixed samples were sectioned to 150–250 µm sections using a vibratome (Leica), and then permeabilized and blocked with 0.1% Triton X-100 solution and 10% donkey serum in PBS for 30 min at room temperature. Then primary antibody (Apo-B; Abcam, ab20737) was added (1:100 in 1.5% BSA/PBS solution) and the samples were incubated overnight at 4 °C, followed by multiple PBS washes. Cells were then incubated with secondary fluorescent antibody (Invitrogen, SA5-10038) and phalloidin (Invitrogen, A12380">A12380) at room temperature for 60 min and washed with PBS; nuclei were co-stained with Hoechst 33342 (Sigma, 14533). Microscopy was performed with a laser scanning confocal microscope (Leica SP5 X MP DMI-6000 or Zeiss TIRF/LSM 710).

For the measurement of neurite length, the cells were seeded on cover glasses at a density of 1 × 10⁵ cells per 12-well plate. Subsequently, transfection and immunostaining of the cell were performed following standard procedures. The cell edge was determined with phalloidin (Invitrogen #A12380) staining.

The cortical cytoskeleton imaging experiments were performed on Hela cells. Hela cells were seeded in a 35-mm confocal dish 24 h before experiments, and then the cells were treated with 10 mM MβCD or 10 μM 3oc for 0, 10, 30, 60, 120, and 240 min. After the cells were fixed, Phalloidin-Alexa Fluor 568 (1:200, Invitrogen, A12380) and DAPI (1:1,000) were used to stain the cytoskeleton and nucleus separately. Finally, the confocal imaging was performed, and ImageJ was used for the statistical analysis of average fluorescence intensity.
The 3D imaging of GPMVs was performed at room temperature by a commercial Spinning Disk Confocal Microscope (Andor Dragonfly, Oxford Instruments) equipped with a 100 × 1.45 NA objective. During imaging, DiD was excited by a 647-nm laser, and the emission was collected by using a 660–700 nm band-pass filter. The 3D imaging was recorded with 2,048 × 2,048 pixels and with a step size of 200 nm. The two-dimensional (2D) and Z-stack images were processed by ImageJ “Z project” plugin.

Determination of the numbers of each cell type per unit length in microdissected PTs and CCDs from UNx and Sham mice was carried out using immunocytochemistry employing antibodies recognizing cell type specific markers, based on Purkerson et al.^{79 (link)}. The primary antibodies used were rabbit anti-AQP1 (LL266, in house, 1:100), mouse anti V-ATPase B1/B2 (F-6, sc-55544, Santa Cruz Biotechnology, Santa Cruz, CA, 1:100), anti-chicken AQP2 (CC 265, in house, 1:1000) and Alexa Fluor 568 phalloidin (A12380, Invitrogen, 1:400). The secondary antibodies were Alexa Fluor 488 goat anti-rabbit, Alexa Fluor 488 goat anti-chicken, Alexa Fluor 568 goat anti-chicken and Alexa Fluor M-594 goat anti-mouse IgG. (A11034, A11039, A11041 and A11032, Invitrogen) each at 1:400 dilution. Cell nuclei were labelled with DAPI. Confocal fluorescence images were recorded with a Zeiss LSM780 confocal microscope using a 20× objective lens by Z-stack scanning. 3D images are reconstructed using z-stack files, and cell counting was performed on three-dimensional reconstructed tubule images using IMARIS Scientific Image Processing & Analysis software (v7.7.1, Bitplane, Zurich, Switzerland). Counting was automated using IMARIS “spot analysis” for nuclei. Tubule volume was calculated using IMARIS “surface analysis”.

P1 staining: eight wandering 3rd instar larvae from each group were bled in 10 μL of PBS, and then hemocytes were transferred to an adhesive glass slide. After incubation at room temperature for 30 min, hemocytes were fixed with 4% PFA, blocked with PBST containing 5% goat serum and incubated with anti-P1 (a gift from Professor István Andó) at 4 °C overnight. The next day, hemocytes were incubated with Alexa Fluor 568-conjugated secondary antibodies (A-11004, Invitrogen) and Hoechst dye for 2 h and 10 min, respectively, and mounted with Slowfade.
F-actin staining: eight wandering 3rd instar larvae were bled in 10 μL of PBS, incubated at room temperature for 30 min, and fixed with 4% PFA for 10 min. After 3 washes, hemocytes were incubated with Alexa Fluor 568-conjugated phalloidin (A12380, Invitrogen) and Hoechst dye for 30 min and 10 min, respectively. Then, hemocytes were mounted with Slowfade.
TUNEL and 7-AAD staining: To examine cell apoptosis and death, hemocytes were incubated with a TUNEL kit (11684795910, Roche, Basel, Switzerland) and 7-AAD (5 μg/mL, A1310, Invitrogen), respectively, according to the manufacturer’s instructions.
All stained sections were analyzed under a fluorescence microscope (Axio Scope A1, Zeiss, Germany).