P1187 Exp. 6Confocal Study: Branfman Project 2/18//03Description: alpha-synuclein immunolabeling for large-scale mapping study (non-tg)Animals: 548 (f) transgenic from D-line (see subject log for details)Protocol1. PerfusionNembutal; 4% paraformaldehyde + 0.1% gluteraldehyde1 hr. postfix in 4% para2. Sectioned on VibratomeThickness = 80 microns3 blocks at 2 mm each from anterior (A, B, C) + cerebellumLeft hemisphere marked3. Wash 6x with PBS 1X (on ice)4. Make up blocking bufferPBS w/o NaCl = buffer usedTotal amount needed = 33 mls x 2IngredientAmount0.8 PBS6.6 ml 5X PBS + 24.2 ml 2x distilled H203% NGS (24 , 7 )0.96 ml1% fish gel0.33 ml0.1% Triton X-1000.0332 ml1% BSA0.33 g5. Block slices (1 hr) in blocking bufferIngredient Amount0.8 PBS 6.6 ml 5X PBS + 24.2 ml 2x distilled H203% NGS (24 , 7 ) 0.96 ml1% fish gel 0.33 ml0.1% Triton X-100 0.0332 ml1% BSA 0.33 g6. Make up working bufferUse blocking buffer to dilute to working bufferIngredientAmountBlocking buffer20 ml0.1% Triton0.2 ml1X PBS180 ml7. Wash 1X5 minutes with working buffer 8. Dilute primary Abs in working bufferanti-alpha-SYN; Host = Rabbit 1:5009. Place on shaker in cold room labeled & covered with aluminum foil overnight10. Wash 6x with working buffer11. Prepare secondary Abs (for confocal immunolabeling)goat anti-rabbit - AF 568@ 1:50 12. Let sit on cold room shaker covered with foil for 48 hrs13. Wash 3x with 1X PBS 0.814. Prepare nuclear stain* Final solution = equal parts 2xPBS + 1:100 Hoescht 33342 in ddH2O* Prepare each separately. * Once added together, you should not observe any precipitation.* If precipitation is observed.... Do not use the solution!15. 30 min staining with nuclear stain16. Wash 3x with 1X PBS 0.817. Sections mounted on slides and coverslipped using Gelvatol
Imaging product type
Type
Optical section
Cutting plane
transverse
Z resolution
2.5 um
Citation Information
Diana Price, M.H. Ellisman; M. Martone; G.A. Johnson; E. Masliah (2002) CCDB:29, mus musculus. CIL. Dataset. https://doi.org/doi:10.7295/W9CCDB29