Rabbit anti pten

Sections were stained using the Bond RX autostainer (Leica Biosystems Inc., Buffalo Grove, IL, USA). Briefly, slides were baked at 65 °C for 15 min and the automated system performed dewaxing, rehydration, antigen retrieval, blocking, primary antibody incubation, post primary antibody incubation, detection (DAB), and counterstaining using Bond reagents (Leica). Samples were then removed from the machine, dehydrated through a series of ethanol and xylenes and mounted. Rabbit anti-γ-H2AX (Ser139) (1:800, #9718, Cell Signaling), rabbit anti-phospho-EGFR (Tyr1068) (1:50, #2234, Cell Signaling), rabbit anti-phospho-HER2/ErbB2 (Tyr1221/22) (1:400, #2243, Cell Signaling), rabbit anti-PTEN (1:150, #9559, Cell Signaling: used for mouse transplants and normal reduction mammoplasty samples), mouse anti-PTEN (1:100, #M3627, Dako: used for HER2 breast cancer patient cohort), rabbit anti-Ki67 (1:200, #ab16667, Abcam) were diluted in antibody diluent (Leica). TUNEL staining was performed using manufacturer’s recommendations (ApopTag Peroxidase In Situ Apoptosis Detection Kit, #S7100; EMD Millipore). All microscopic imaging was done using the VECTRA^® Automated Quantitative Pathology Imaging system or an Axioskop 40 with ZEN Software (Zeiss, Germany). Whole tissue imaging was done using a Stemi SV 11 Stereoscope with ZEN Software (Zeiss).

The following IHCs, according to manufacturers’ protocols were performed: Freshly isolated tissues were fixed with formalin and embedded with paraffin, cut into 4–5 µm slices, and fixed on slides with a polylysine coating. The slides were deparaffinized in xylene followed by graded ethanols (100%, 95%, 70%, 50%), and finally washed in cold running water. Endogenous peroxidase was removed using 0.5% hydrogen peroxide in methanol solution, and antigen blocking was carried out using 5% bovine serum albumin, following antigen retrieval in a pressure cooker using a thermally-induced method that the slides were put into an autoclaver containing medlar buffer, heated for 5 minutes, and then cooled in cold water to repair epitopes in citrate buffer (pH 6). Slides were incubated with primary antibodies at different dilution ratios to determine the optimal concentrations. The slides were then washed and treated with a secondary antibody. Incubations with antibody (Rabbit anti-PTEN, Cell Signalling, Massachusetts, USA) was carried out in a humidified box to avoid drying. After incubation with the secondary antibodies, the slides were washed with PBS, covered with freshly prepared DAB color solution, and then washed again with water, followed by hematoxylin staining, clearing, drying, and mounting. The immunohistochemical stains were evaluated by two pathologists with consensus.

Mice were transcardially perfused with phosphate buffered saline followed by 4% paraformaldehyde (ThermoFisher). Once dissected, tissue was postfixed overnight in 4% paraformaldehyde, then overnight again in 20% sucrose (ThermoFisher) in 0.1 M phosphate buffer. The tissue was then frozen in Cryomatrix (ThermoFisher) and sectioned at 20 μm (DRG and sciatic nerve) or 50–100 μm (spinal cord). In some cases (Fig. 1), whole sympathetic or sensory ganglia were stained and imaged. All sections were blocked in 10% normal donkey serum with 0.2% Triton X-100 plus 0.02% sodium azide in PBS. Sections were incubated overnight with primary antibodies: rabbit anti-ATF3 (1:400, Santa Cruz SC-188), rabbit anti-ATF3 (1:500, Novus NBP 1-85816), rabbit anti-SCG10 (1:1000, Novus NBP 1-49461), rabbit anti-PTEN (1:400, Cell Signaling 9188), and mouse anti-PTEN (1:200, Cell Signaling 14642). Sections were incubated for 2 h with the appropriate secondary antibodies at a concentration of 1:1000: Alexa Fluor 488 donkey anti-rabbit (Invitrogen A21206), and Alexa Fluor 488 donkey anti-mouse (Jackson ImmunoResearch 715-545-151). Slides were cover-slipped with ProLong Gold with DAPI (Invitrogen).

Aorta were homogenized in RIPA buffer. Denatured protein (50 μg) were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA) were blocked in Tris-buffered saline buffer with skimmed milk for 30 min, followed by overnight incubation at 4 °C with rabbit anti-PTEN (1:1,000, Cell Signaling Technology, Danvers, MA, USA) or mouse anti-Bcl-2 (1:1,000, Abcam, Cambridge, UK). After washing, membranes were incubated with horseradish peroxidase conjugated secondary antibody for 2 h at room temperature. After incubation, membranes were washed and developed using a chemiluminescence (ECL, Cell Signaling Technology, Danvers, MA, USA) assay. The housekeeping protein β-actin (1:5,000, Abcam, Cambridge, UK) was used for normalization.