Axiovision

Whole embryos were imaged in 3% methylcellulose using a Zeiss Stemi SV 11 microscope with an AxioCam HRc (Zeiss) camera and AxioVision (Zeiss) software. For sectioned samples, embryos were dehydrated with an ethanol work-up and embedded in JB4 medium (Polysciences Inc.) and 7-μm-thick sections were cut with a Leica microtome. Sections were collected on glass slides and mounted with a coverslip in Aquapolymount (Polysciences Inc.) and imaged using a Zeiss Axioplan2 microscope with an MRc camera (Zeiss) and AxioVision (Zeiss) software.

1×10⁵ RK13 cells were infected with 1×10⁶ spores on glass coverslips imprinted with three location markers. Cells were fixed with 4% paraformaldehyde in PBS for 30 min after three days post-infection. After being washed three times by TBST (TBS plus 0.05%Tween 20), coverslips were blocked by 3% BSA in TBST for 1hr at room temperature. EhPTP4 monoclonal antibody (MAb-EhPTP4; clone F4-6) at a 1:10 dilution and EhPTP1 rabbit polyclonal antibody (rab-PcAb-EhPTP1) at a 1:500 dilution were incubated with cells for 1hr at room temperature. After being washed three times by TBST, Alexa Fluor 488-labeled anti-mouse IgG antibody and Alexa Fluor 594-labeled anti-rabbit IgG antibody were added at 1:500 dilutions. Mouse and rabbit preimmunization sera were used as negative controls. Samples were washed three times by TBST and imaged using a Zeiss AxioObserver microscope equipped with Axiovision software with "shuttle & find" to mark cell locations. After fluorescence imaging, samples were fixed with 2.5% glutaraldehyde, dehydrated in ethanol, critical point dried (Tousimis Samdri 790), and coated with chromium (EMS 150T-ES). The same cells were automatically located in the Zeiss Supra 40 Field Emission Scanning Electron Microscope and imaged with a secondary electron detector. Fluorescence and SEM images were correlated with Zeiss AxioVision.

For histology, 5 µm paraffin sections were stained with Hematoxylin and Eosin (H&E) as previously described^{6 (link)}. Mosaics of H&E-stained kidney sections were created under a 10x lens using a motorised Zeiss Axiovert200 microscope equiped with a color camera AxioCam HRc and Zeiss AxioVision software (Zeiss, Oberkochen, Germany). Zooms were taken under a 40x lens. The degree of tubular dilation was automatically quantified using a Nikon digital camera Dx/m/1200 and NIS software (Laboratory Imaging Ltd). Ten randomly selected microscopic fields (X200) were scored. Surgical scars were excluded from analysis.
For immunostaining, 8μm (cilium and centrosome analyses) or 30 µm (mitosis analyses) paraffin sections were dewaxed in a xylene bath and rehydrated once in isopropanol and in increasing ethanol solutions. Sections were saturated in 10% goat serum (Dako, Carpinteria, California, USA) for 30 min. Primary antibodies incubations were performed at 4 °C for 12 h in 10% goat serum solution. Secondary antibody incubations were done at room temperature for 2 hours in 10% goat serum solution. Tissue sections were mounted in Mowiol 4-88 (EMD Millipore) solution. To label proliferative cells, paraffin sections were stained with the Click-iT Edu Alexa Fluor 488 Imaging kit (Thermo Fisher Scientific).

GSCs were isolated as previously described [17 (link)]. Briefly, GBM cells were cultured in the appropriate medium for isolation of GSCs, consisting of DMEM/F12 supplemented with 20 ng/mL epidermal growth factor (EGF) (R&D Systems, Minneapolis, USA), 10 ng/mL fibroblast growth factor-2 (bFGF) (PeproTech Inc., Rocky Hill, NY, USA), and 1% penicillin/streptomycin. Cells were cultured in a humidified incubator with 5% CO₂, 5% O₂ at 37°C, and split once a week. At each passage (P), GSC neurospheres were dissociated with Tryple Select (Gibco, Grand Island, NY, USA) and Trypan Blue dye exclusion assay (Gibco) was used to assess cell viability. Cells were routinely observed with an inverted phase-contrast microscope (Nikon Eclipse TE300; Nikon, Shinjuku, Tokyo, Japan), and images were acquired with a digital camera (Zeiss Axiovision, Zeiss, Oberkochen, Germany).