Rabbit psd95

Membranes were transferred into 6 well plates and slices were fixed for 20mins in 4 % paraformaldehyde in 0.1 M PBS (applied both above and below the membrane insert). To reduce the volumes required for subsequent steps, the membranes were cut free of the plastic inserts and the sections of membrane containing the slices were transferred, using forceps, to individual wells in a 24 well plate. Slices were washed twice in TBS, blocked for 1 h in blocking solution (TBS with 0.5 % Triton X-100 and 3 % Goat Serum) then incubated in 200 μl primary antibody diluted in blocking solution overnight at 4 °C with shaking. Slices were washed 3 times in TBS before being incubated (2 hs, RT in the dark) with Alexa488, 568 or 647 conjugated secondary antibodies (Life Technologies-diluted 1:250 in blocking solution). After a final 3 TBS washes, some slices were counterstained with Thioflavin S, BTA-1, Nissl or Hoechst. Images were captured using a Nikon Confocal Microscope. Primary antibodies used: mouse Tuj1 (Covance 1:1000), rabbit Tuj1 (Sigma 1:500), chicken Tuj1 (Abcam:1:1000) rabbit NFL (Millipore 1:250), mouse MOAB2 (pan specific to Aβ- Millipore 1:1000), rabbit GFAP (Abcam 1:1000), mouse synaptophysin (Dako 1:1000), rabbit PSD95 (Abcam 1:500), rabbit tau (Dako 1:1000), rabbit Iba1 (Wako 1:500) rabbit calbindin and rabbit parvalbumin (Kind gifts from Dr P Emson 1:1000).

Slices were scraped off the membrane, treated with 2x Laemelli buffer + 10 % 2-mercaptethanol (250 μL per 3 slices) vortexed, boiled, then frozen at -20 °C until use. For use, most samples were further diluted 1:2 and loaded 8 μl per lane in a precast 4-20 % gradient gel (Bio-rad). To detect the weaker signal for PSD95, 15 μl of undiluted sample was loaded. After incubation in primary antibody overnight, blots were probed with 1:5000 mouse-700 (Life Technologies) and rabbit-800 (LI-COR) secondary antibodies then imaged using a LI-COR Odyssey detection system. Band intensity (IKK) was quantified using Odyssey software then normalised to Tuj1 signal. Primary antibodies used: mouse synaptophysin (Abcam: 1:1000), rabbit tuj1 (Sigma: 1:2500), rabbit PSD95 (Abcam: 1:500), mouse VAMP2 (Synaptic Systems: 1:10,000) and mouse RT97 (Kind gift from Dr Diane Hanger 1:500). Tuj1-normalised protein expression was then compared between WT and TgCRND8 cultures, TgCRND8 values expressed as a percentage of the WT average.