Jefferis Lab DokuWiki si:kohl2014

si:kohl2014:fiber_analysis_in_mouse_brain_slices

Anonymous (anonymous@undisclosed.example.com) — 2014-07-21T16:27:25+00:00

200 µm coronal vibratome brain slices of animals injected with AAV-myrSNAP were incubated with 1 µM BG-549 for 30 min, 1 h, 6 h or 12 h. Confocal stacks were resliced and individual neurites that projected through the whole depth of the stack (n = 9–11 per condition) were traced using the ImageJ Simple Neurite Tracker tool; the tracing paths were converted to a binary mask by thresholding the result of the

si:kohl2014:fly_image_analysis

Anonymous (anonymous@undisclosed.example.com) — 2014-07-21T16:29:43+00:00

For quantification of substrate penetration into fly brains, samples from GH146-Gal4 > myrSNAP or GH146-Gal4 > myrHalo or brp-SNAP animals were incubated with 1 µM BG-549 or 1 µM Halo-TMR for 15 min, 30 min, 1 h, 4 h or 12 h. Samples from GH146-Gal4 > myrGFP or Canton-S animals were incubated with anti-GFP (1:1,000) or anti-nc82 (1:30) antibodies for 15 min, 30 min, 1 h, 4 h or 12 h, followed by washing and incubation with fluorescent secondary antibodies (anti-chicken Alexa 568 and anti-mouse …

si:kohl2014:start

Anonymous (anonymous@undisclosed.example.com) — 2014-11-13T20:23:53+00:00

Please see main tagging page

si:kohl2014:tagging2014fly-cloning

Anonymous (anonymous@undisclosed.example.com) — 2014-04-01T12:43:22+00:00

Drosophila constructs * pTW, pTW, pTW and pTW constructs were made using the Multisite Gateway technology platform (Invitrogen). CLIPf and SNAPf are engineered versions of the original versions CLIP26m (CLIPm) and SNAP26m (SNAPm), respectively, that display faster labeling kinetics [Sun:2011dq, Pellett:2011oq]. pTW is a pUASt vector containing a Gateway cassette. All CLIPm, CLIPf, SNAPm and SNAPf coding sequences were amplified from pCLIPm, pCLIPf, …

si:kohl2014:tagging2014flystain

Anonymous (anonymous@undisclosed.example.com) — 2014-04-01T13:54:07+00:00

Protocol for chemical labeling of Drosophila brains non cell-permeable substrates (recommended) * dissect brains in ice-cold 0.1 M PB * fixation in 4% PFA (in 0.1 M PB) at room temperature for 20 min in glass-well plate on nutator * SNAP-tag / CLIP-tag: no change in labeling intensity after prolonged fixation of up to 60 min.

si:kohl2014:tagging2014mousestain

Anonymous (anonymous@undisclosed.example.com) — 2014-04-01T13:19:22+00:00

Protocol for chemical labeling of mouse brain slices Vibratome slices (200 µm) * fix slices in 4% PFA (in 0.1 M PB) at room temperature for 20 min in glass-well plate on nutator * remove fixative and and wash 2 x 5 min with PBT (0.3% Triton X-100)

si:kohl2014:tagging2014recflies

Anonymous (anonymous@undisclosed.example.com) — 2014-04-01T14:46:52+00:00

Recommended fly stocks for chemical labeling see full list chemical labeling fly stocks membrane labeling SNAP * {myrSNAP}attP40 or {myrSNAP}attP2 better signal in neurites * CD4-SNAP/CyO;CD4-SNAP/TM6b stock enriched in cell bodies CLIP * CD4-CLIP#1/CyO no myrCLIP available at the moment

si:kohl2014:tagging2014recommend

Anonymous (anonymous@undisclosed.example.com) — 2014-04-01T13:26:59+00:00

si:kohl2014:tagging2014substrates

Anonymous (anonymous@undisclosed.example.com) — 2014-04-01T14:56:25+00:00

Notes on substrates for chemical labeling * store substrates at -20°C in presence of dessicant this avoids hydrolysis of substrates * for substrates that are purchased in dry powder form: dissolve in fresh, water-free DMSO using fresh DMSO from a sealed container is crucial as DMSO is hygroscopic; we observed reduced labeling efficiency when using substrates that were dissolved in old DMSO, presumably due to substrate hydrolysis

si:kohl2014:tagging2014virus-cloning

Anonymous (anonymous@undisclosed.example.com) — 2014-04-01T12:53:49+00:00

* pAAV * pAAV * pAAV