I'm using a Rosa-YFP reporter mouse to check Cre expression. We seem to be getting a fair amount of autofluorescence, and we're trying to troubleshoot. Any tips or suggestions about the perfusion, reagents, handling of the brains, etc would be most helpful. Thanks!
2007-08-25 17:02:22 · 2 answers · asked by Istra 3 in Science & Mathematics ➔ Biology
we're perfusing the mice with ice cold 4% PFA, then fixing them in 4% PFA for 24 hours, then move to 30% sucrose for 48 hours, then section at 35 um on a sled microtome. then they are dapi treated (i've also used an anti-GFP antibody and then dapi, which definately enhanced my ability to distinguish real signal from autofluorescence) and mounted on a + charged slide, then vectashield and coverslip sealed with clear nail polish. i'm looking at the slides within 2 days of their mounting. we're using a FITC filter on our scope, though we checked 2 other labs scopes with different GFP filters and they looked the same. when i look at the slides, theres a background level of fluorescence (i know its not antibody background because its also in the sections not treated with antibody). in areas that are strongly positive for YFP, i can visually tell the difference between the autofluorescence and the real thing, but there are some questionable areas i'm still trying to resolve. thanks again.
2007-08-26 04:24:05 · update #1
What did you do for detection? How did you process your samples? It is difficult to give meaningful suggestion without knowing what did you do. For example, did you fix your samples? Did you process your sample for FACS or microscopic examination? Usually, YFP is not as bright if you fix the sample with alcohol based method. PFA works better. Did you use tissue for frozen slides or paraffin embedded slidcing? How long after fixation and processing did you observe your slides? What is the light source? What is the emmision and exciting weave length did you use? What is the results look like?
Maybe you have read the following article already, they had nice results:
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WDG-4H6PKTP-2&_user=10&_coverDate=11%2F01%2F2005&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=42916f502d1c73a01bada52b17cfa485
2007-08-25 18:25:15 · answer #1 · answered by Dolorsas 2 · 0⤊ 0⤋
YFP requires no co factors to fluoresce, so either the reporter you're using floats around or some perfusion reagent you're using is fluorescing.
2007-08-25 17:11:29 · answer #2 · answered by BP 7 · 0⤊ 0⤋