Ba 1000

O.C.T.-embedded tissue slides were prepared and stored at − 20. Slides were thawed at room temp (RT) for 30 min, and incubated at − 20 in acetone for 10 min, followed by air drying (10 min). 1× PBS rinse and Avidin block (Vector labs, Burlingame, Blocking kit cat #sp-2001) for 10 min were performed. Three minutes 3× PBS wash and 10 min Biotin block at RT were performed. 3× PBS wash followed by serum-free protein block (Dako cat # 2013-09) for 30 min at 37 °C was performed. Cleaved Caspase 3 antibodies (cell signaling #9661S) were diluted (1:100) in Dako antibody diluent (Cat #S202230-2CN) and incubated with tissue samples overnight at 4 °C. 3× PBS washes were followed by biotinylated anti-rabbit (Vector Labs Cat #BA-1000) 1:300D for 30 min at RT, 3× PBS washes and 1:300 cy3-streptavidin (Vector Labs cat #BMK-2202) for 30 min at RT, and 3× PBS washes and final wash with Vectashield (Vector Labs, Cat #H-1200) with DAPI followed by storage at 4 °C. Immunofluorescence signal was quantified by ImageJ (n = 3 representative images per condition), and CC3 signal normalized to corresponding DAPI channel, and statistical significance determined using Student’s t test.

We fixed nerve explants in 4% FA (formaldehyde) followed by preparation of 5 μm paraffin microtome slices. Immunohistochemistry was performed using Biotin conjugated secondary antibody anti-rabbit (1:500; BA-1000, Vectorlabs) and a peroxidase based detection system using the ABC kit (PK-6100, Vectorlabs) and DAB as substrate. Alternatively, Alexa488 or 546 (1:500; anti-rabbit 488, A-11008; anti-mouse 546, A-11003, Thermo Fisher Scientific) conjugated secondary antibodies were used. Primary antibodies included anti-S100β (rabbit, 1:1000, ab52642, Abcam), anti-βIIITub (mouse, 1:3000, MMS-435P-200, Eurogentec) and anti-cJun (rabbit, 1:500, #9165, Cell signaling).
For teased fibres, murine sural nerves were fixed in 4% FA overnight, teased using forceps and dried on glass slides. Staining was performed using primary antibody anti-Pparg (rabbit, 1:200, ab45036, Abcam) and secondary antibody Alexa488 (1:500; anti-rabbit 488, A11008, Life Technologies).

Tumor tissues from flank tumors and whole brain tissues from P7, adult Ptch1^+/+Trp53^-/-, and adult Ptch1^+/-Trp53^-/- mice were fixed in 4% formaldehyde overnight and standard paraffinization was performed. Sections were cut to 5 um thickness. Sections were rehydrated in xylene and serial ethanol concentrations. Antigen retrieval was achieved with sodium citrate at sub-boiling temperature in the microwave for 30 minutes. Sections were incubated overnight at 4°C with Foxo3a antibody (Cell Signaling, D19A7) or hexokinase 2 antibody and subsequently with anti-rabbit biotinylated secondary antibody (Vector Labs, BA-1000) for 30 minutes at room temperature. For hexokinase 2, sections were incubated with avidin/biotin ABC complex (Vector Labs, PK-6102) and stained with DAB substrate chromogen (DAKO, 2016–10). For Foxo3a, after secondary antibody incubation, sections were incubated with fluorescein-labeled avidin D for 30 minutes (Vector Labs). Slides were then mounted with Vectashield DAPI mounting medium for immunofluorescence (Vector Labs). High magnification images of sections were captured using a Nikon 90i microscope equipped with Roper EZ monochrome and DS-Fi1 color cameras. NIS Elements BR 3.0 software was used to capture and analyze the images.

ELISA plates (Multiwell Immuno Plate, Maxisorp, 96 well; M9410, Thermo Scientific) were coated with 8 μg/ml goat anti-mouse Gal1 capture antibody (AF1245, R&D Systems) in PBS pH 7.4 and blocked with horse serum. Subsequently, the standard (recombinant mouse Gal1; 1245-GA, R&D) or mouse sera (diluted 1:15 in PBS) were added. Rabbit anti-human Gal1 antibody (0.75 μg/ml; 500-P210, Peprotech) was used as detection antibody, followed by 3 μg/ml biotinylated goat anti-rabbit IgG (BA-1000, Vector Laboratories) and 1 μg/ml streptavidin–horseradish peroxidase. Enzymatic detection was performed as described above. A standard curve ranging from 2.5 to 80 ng/ml of recombinant mouse Gal1 was used to calculate Gal1 concentrations in the samples. Four-parameter logistics regression was used for fitting the standard curve.