Mowiol

For immunocytochemistry on cultured cells, cells were seeded on PDL-coated coverslips in a 24-well plate at a density of 2 × 10⁴ cells. After three days in culture, the cells were fixed in 4% paraformaldehyde (PFA) dissolved in phosphate buffer saline (PBS), pH 7.4 for 30 min. Cells were washed in PBS, incubated in a blocking buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.25% (w/v) gelatine, and 0.5% triton X-100) at room temperature for 15 min, and afterwards with primary antibodies (Supplementary Table 2) in blocking buffer overnight at 4 °C. Coverslips were washed with PBS and incubated with secondary antibodies (Supplementary Table 2) and Hoechst 33528 (1:1000, Thermo Fisher Scientific, H3569) in blocking buffer at room temperature for 1 h. After washing steps with PBS, the coverslips were mounted on microscopy slides with Mowiol (0.1 M tris–HCl pH 8.5, 25% glycerol, 10% Mowiol (Merck Millipore, 81381). The samples were imaged using a Zeiss Axioscope A1 microscope with a 40 × objective.

To perform immunocytochemical staining, cells were cultured on uncoated glass coverslips, fixed with 4 % Paraformaldehyde (PFA), washed in PBS, and incubated in SuMi buffer for 10 min. Primary antibodies were diluted in SuMi and incubated at 4 °C on a shaker O/N. Cells were washed 3 times in PBS and, subsequently, incubated with secondary antibodies and Hoechst 33258 (1:1000 dilution) (Invitrogen) diluted in SuMi at RT for 1 h. The antibodies used are listed in Table 2. All secondary antibodies were from Jackson Immune Research and diluted 1:1400 in SuMi. Cells were washed again in PBS, before the coverslips with cells were mounted on slides with Mowiol [0.1 M Tris–HCl pH 8.5, 25 % glycerol, 10 % Mowiol (Calbiochem, Merck Millipore)]. The actin network was visualized with acti-stain Phalloidin 670 (Cytoskeleton inc; 1:1000 dilution). All fluorescent images were taken with a Leica SP5 confocal microscope (Leica) with a 63x objective.Table 2

Primary antibodies

Antibody	Manufacturer	Dilution	Cat #
Pan GFAP Dako	Dako	1:4000 (1:8000 WB)	Z0334
hGFAPδ	Manufactured in house (10-05-2001 Bleed)	1:1000 (1:1300 WB)	–
GFAP c-term	Santa Cruz	1:4000 (WB)	Sc-6170
Vimentin	Chemicon	1:3000	AB5733
GAPDH	Abcam	1:4000 (WB)	AB14247

GFAP glial fibrillary acidic protein, WB western blot, c-term carboxy terminal, hGFAP human GFAP, GAPDH glyceraldehyde-3-phosphate dehydrogenase

Immunohistochemical labelling was always performed on three slices from each mouse, at approximately 25%, 50% and 75% of the distance along the anteroposterior axis of the olfactory bulb. Free‐floating slices were incubated at room temperature (RT; 18–24°C) for 2 h in a blocking and permeabilising solution (10% normal goat serum in PBS containing 0.25% Triton‐X and 0.02% sodium azide). They were then incubated in primary antibody diluted in the blocking solution at 4°C for between 18 and 24 h (see Table 1 for antibodies used and their working dilution; endogenous tdTomato fluorescence was always enhanced with immunolabel for red fluorescent protein; mouse anti‐TH antibody was used for all staining except when TH was co‐labelled with DDC in Figure 1b, in which case rabbit anti‐TH was used to avoid host species cross reactivity). For immunofluorescence intensity analysis, slices were incubated for approximately 72 h in primary antibody solution. Slices were then washed with PBS (3 × 5 min) and incubated in the species‐appropriate secondary antibody (Alexa Fluor® Life Technologies) diluted 1: 1,000 in the blocking solution at RT for 2 h. After 3 × 5 min washes in PBS, slices were mounted on glass slides with MOWIOL® (Millipore).

Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 20 min at room temperature and permeabilized with 0.5% saponine (Sigma-Aldrich) in PBS. Following incubation in 5% bovine serum albumin (BSA) with 0.1% saponine for 20 min at room temperature to block unspecific protein–protein interactions, specific cellular proteins were detected using anti-AKT (pan; Cell Signaling; 2920; 1:50), anti-phospho-AKT (Ser473; Cell Signaling; 4060; 1:400) and secondary antibodies coupled with Alexa Fluor 488 (Thermo Fisher Scientific; A11001; 1:500) or Alexa Fluor 568 (Thermo Fisher Scientific; A11011; 1:500). DNA was stained with DAPI (1 μg/mL; Sigma-Aldrich) for 20 min and cells were mounted in Mowiol (Millipore) and DABCO (Sigma-Aldrich) medium. Fluorescence was recorded using an Eclipse Ts2R microscope (Nikon, Amsterdam, The Netherlands).

Following fixation with 4% paraformaldehyde for 30 minutes at room temperature, MC-38 cells were washed twice with PBS, permeabilized with 0.1% saponin, free aldehyde groups quenched with 20 mM glycine, and unspecific binding sites blocked with 10% FCS for 30 minutes. Proteins were detected by incubation for 1 hour at 37°C with anti-p65/RelA (Santa Cruz Biotechnology) or anti-IκBα (Cell Signaling Technology) antibodies. After washing with PBS, the respective secondary antibodies labeled with Cy3 (Jackson Immunoresearch, West Grove, PA, USA), Alexa488 or Alexa568 (Molecular Probes, Carlsbad, CA, USA) were applied, and nuclei were counter-stained with DAPI. Samples were mounted using Mowiol (Millipore, Darmstadt, Germany) and analyzed by fluorescence microscopy. Spheroids were fixed in 10% formalin for 30 minutes at room temperature and processed for histological analysis using previously described techniques [43 ]. Dewaxed paraffin sections (5 μm) were rehydrated, blocked with 10% normal goat serum and processed as described above.