Large scale brain mosaic at the level of the anterior hippocampus showing the localization of alpha synuclein (green) in a normal mouse. Section was counterstained with a nuclear stain (blue) to reveal the locations of cell somata. Full resolution image may be viewed by clicking on the Neuroinformatica link (N) next to the thumbnail.
P1187 Exp. 10A 2-photon Study: Branfman Project 7/28/03Description: alpha-synuclein + nuclear stain for large scale mapping study in striatal regionProtocol1. Perfusion Nembutal; 4% paraformaldehyde 2 hr. postfix in 4% para2. Sectioned on Vibratome Thickness = 80 microns 3 blocks at 2 mm each from anterior (A, B, C) + cerebellum3. sections stored in cryoprotectant at -20 (7/30/03)4. Wash 6x with PBS 1X (on ice)5. Make up blocking buffer PBS w/o NaCl = buffer used Total amount needed = 33 mls x 3 For each 33 ml: Ingredient Amount 0.8 PBS 6.6 ml 5X PBS + 24.2 ml 2x distilled H20 3% NDS ( 24 , 4) 0.96 ml 1% fish gel 0.33 ml 0.1% Triton X-100 0.0332 ml 1% BSA 0.33 g6. Block slices (1 hr) in blocking buffer7. Make up working buffer Use blocking buffer to dilute to working buffer Ingredient 200ml 150ml 100ml 50ml Blocking 20 ml 15 ml 10 ml 5 ml buffer 0.1% Triton 0.2 ml 0.15 ml 0.1 ml 0.05 ml 1X PBS 180 ml 135 ml 90 ml 45 ml8. Wash 1X5 minutes with working buffer9. Dilute 1o Abs in working buffer anti-alpha-synuclein; Host = Rabbit; 1:500 = 4 microliters in 2 ml WB Vial Contents/Tx Total Volume Amt Ab added/vial 1. a-synuclein 2 ml 4 + 2 ?l 2. Control 2 ml Omitted 4 microliters x 2 vials= 8 x 3 animal = 24 microliters total alpha-synuclein10. Place on shaker in cold room labeled & covered with aluminum foil overnight11. Wash 6x with working buffer12. Prepare 2o Abs (for confocal immunolabeling) donkey a.....rabbit AF488 @ 1:100 (MBIRN Box 5)13. Let sit on cold room shaker covered with foil for 2-24 hrs14. Prepare nuclear stain a. Final solution = equal parts 2xPBS + 1:100 Hoescht 33342 in ddH2O b. Prepare each separately. c. Once added together, you should not observe any precipitation. d. If precipitation is observed?. Do not use the solution! e. 2 ml x total number of vials = total ml solution needed15. 30 min staining with nuclear stain16. Wash 6x with 1X PBS 0.8
Imaging product type
Type
Optical section
Z resolution
2.5 um
Citation Information
Diana Price, M.H. Ellisman; M. Martone; G.A. Johnson; E. Masliah (2002) CCDB:36, mus musculus. CIL. Dataset. https://doi.org/doi:10.7295/W9CCDB36
