BDNF

Prof.Rosa Di Liddo , Dr. Thomas Bertalot, Dr. Sandra Schrenk University of Padova Department of Pharmaceutical and Pharmacological Sciences See western blot protocol on Western Blotting AB-82598 (Protocol of Prof.Rosa Di Liddo,Dr.Thomas Bertalot, Dr. Sandra Schrenk) University of Padova Department of Pharmaceutical and Pharmacological Sciences Sample preparation Whole protein lysate was obtained using RIPA lysis buffer supplemented with protease inhibitors. All procedures were performed on ice and homogenization of samples was executed using a 22G needle syringe. 1. SDS-PAGE Electrophoresis: Sample loaded: 10μg - Poly-Acrylamide gel at 12% 2. Transfer: 0.45μm PVDF Membrane (Transfer for 80 min at 4°C on ice) -140 Volts and 14 mA 3. Blocking: - Blocking solution: 5% no-fat milk with TBS -Time: 2h 4. Incubation of Primary antibody: Dilution of primary antibody: 1:1000 [1μg/mL] -Dilute buffer: 1% no-fat milk with TBS - Time and temperature: 4°C overnight in constant slow shacking 5. Washing: Wash with TBS-T (Tween-20, 0.25%) for three times (15 min, 5 min, 5 min) 6. Incubation of Secondary antibody: Dilution of secondary antibody: HRP-Goat anti-rabbit IgG (1:2500) 7. Dilute buffer: 1% no-fat milk with TBS -Time and temperature: RT, 90min on shaker 8. Washing: Wash with TBS-T (Tween-20, 0.25%) for four times (20 min, 5 min, 5 min, 5 min) 9. ECL chemiluminescent method:

Immunoflurescence AB-82598 (Protocol of Prof.Rosa Di Liddo,Dr. Thomas Bertalot,Dr. Sandra Schrenk) University of Padova Department of Pharmaceutical and Pharmacological Sciences Procedures 1.Cell culture ENSc were seeded on glass coverslips and maintained in standard medium for 7 days 2. Fixation ENSc were fixed using a paraformaldehyde solution kit for 20 min at 4°C 3. Permeabilization Samples were permeabilized using a 0.5% Triton X-100 solution prepared in PBS 1X for 15 min at RT 4.Blocking Blocking solution: 5% BSA in PBS 1X for 45 min at RT 5. Incubation of Primary antibody Dilution of primary antibody: 1:50 [20μg/mL] Dilute buffer: 0.5% BSA in PBS 1X Time and temperature: 4°C overnight 6. Washing Wash with PBS 1X for three times 7. Incubation of Secondary antibody Dilution of secondary antibody: PE-Goat anti-rabbit IgG (1:100) [10μg/mL]- Dilute buffer: 0.5% BSA in PBS 1X Time and temperature: RT, 60min 8. Washing Wash with PBS 1X for three times 9.Mounting Mount the sample with Fluoromont G with DAPI

IHC(P): Mouse Brain Tissue By Heat: Boiling the paraffin sections in 10mM citrate buffer, pH6.0, for 20mins is required for the staining of formalin/paraffin sections