Dm6 b upright microscope

In situ hybridization was performed using the “BaseScope” method (Wang et al., 2012 (link)) using a kit supplied by ACD (Newark, California, USA), and 15 μm fresh frozen sagittal mouse brain sections. Custom “3ZZ” probes were designed by ACD and were targeted to heme A:farnesyltransferase cytochrome c oxidase assembly factor mRNA (Cox10, GenBank accession NM_178379), nucleotides 788-934 and aldehyde dehydrogenase 1 family, member L1 (Aldh1l1, NM_027406), nucleotides 1256-2112. Aldh1l1 hybridization was used as a marker for astrocytes and was detected by precipitation of Fast Red. Cox10 hybridization was detected by precipitation of Fast Green. Images were acquired with a Leica DM 6B upright microscope and a×40 oil immersion objective using Leica LAS X acquisition software.
Expression was evaluated using ImageJ 1.53c (Schneider et al., 2012 (link)). Cox10 expression was assessed by placing random regions of interest (ROI; defined by a 30 μm circle) over a field that contained at least one red signal suggesting that it was within an astrocyte. Pixels containing blue Cox10 staining were then counted within the ROI. 15 ROIs were evaluated within each 312 μm × 234 μm image from various brain regions. Images were uniformly adjusted for brightness, contrast, and color balance.

HEK293 cells expressing WT- or TKM-5-LOX with or without FLAP were seeded out at 200 cells/ml in each well of an Ibdi 12-well slide. After 24-48 hours at 37 °C 5% CO₂,2.5 μM Ca²⁺-ionophore A23187 was added to stimulate the cells, which were then incubated for 5 minutes at 37 °C 5% CO₂. The cells were fixed with 4% paraformaldye then washed 3 times with PBS. The cells were then incubated with a 50 mM NH₄Cl solution, followed by a wash cycle and subsequently blocked for an hour with 10% donkey serum, 0.1% Tween-20 in PBS. The samples were then incubated overnight at 4°C with a primary antibody solution consisting of either rabbit anti-5-LOX “Body” +/− goat anti-FLAP or rabbit anti-5-LOX “Tail” +/− goat anti-FLAP in 0.1% Tween-20 PBS solution. After a wash cycle with 0.1% Tween-20 PBS the cells were incubated with a secondary antibody solution consisting of donkey anti-rabbit AlexaFluor488 +/− donkey antigoat AlexaFluor647 in a 0.1% Tween-20 PBS solution for 20 minutes. After another wash cycle, the cells were incubated with 2 μg/μl DAPI solution for 3 minutes followed by another wash cycle. Invitrogen Prolong Gold antifade reagent and a coverslip were then added to the slide. The slide was imaged with a Leica DM6B upright microscope using DAPI, GFP (5-LOX), and Cy5 (FLAP) filters.