IHC Test Results: 9/21/12

9/21/2012 Immuno Results (Preliminary)

General: Tissue in tatters, few sections per group, & many sections missing, but everything appeared to work and the sections were mounted in great anatomical register

Short vs Long: Seems clear that the short runs (2 hr in 2º, 90 min in SAvidin) worked as well as the long run (3+ hr in 2º, 2 hr in SAvidin). For the vGAT "blind control", where run conditions were not noted on either slide, I couldn't really tell the difference.

rb ∝ GAD (1: 20, 30 & 40k): All dilutions worked, but (I think) 1:20k was best--more because labeling was good and background was low, so there is little cost to going with 1:20k, rather than some particular gain

rb ∝ DYN (1: 5, 10 & 15k): Could see well-labeled fibers at all dilutions, but no clear concentration of fibers in a recognizable terminal field. Could see lots of labeled cells, somewhat concentration dependent so am not absolutely sure that this is specific labeling. To accurately assess, comparison with DAB series needed to identify the most prominent terminal fields, and the best sections/levels. If not present in these test sections, then the test series will need to be run again

rb ∝ vGAT (1: 2.5, 5 & 10k): Labeling saturated making it difficult to distinguish individual components at 1:2.5k, and it looked as though labeling may have fallen off a bit at 1:10k. Labeling at 1:5k looked great (although I'd bet that 1:7.5k would be optimal). As noted above, labeling group was not recorded on either slide.

Alexa 488●rb ∝ FITC (1: 750, 500 & 2k): Note that the original plan called for 1: 750, 1500 (= 1.5k) & 2k, so only 750 and 2k dilutions mounted. Both worked/looked great. However, at 1:2k there appeared to be some fibers better labeled than others. Did not make comparisons needed to tell whether this was due to concentration, or fiber size (or similar). To make best determination, further comparison of 1:750 & 2k groups needed, as well as comparison of ∝ FITC with SAvidin-labeled NT. However, the 1º has been in solution at 4ºC since July 2010, so I took my best guess and aliquoted the 1º in order to store it at -70ºC.  Aliquots contain 15 µl, which makes 15 ml at 1:1k, or 30 ml at 1:2k