Computational protocol: Effect of dietary fish oil on mouse testosterone level and the distribution of eicosapentaenoic acid-containing phosphatidylcholine in testicular interstitium

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Protocol publication

[…] The slices were fixed with 4% paraformaldehyde for 10 min. After rinsing in phosphate-buffered saline (PBS), endogenous horseradish peroxidase in the tissue slices was blocked using aqueous hydrogen peroxide (a 3% solution in methanol) for 8 min. After rinsing in PBS, the tissue slices were blocked with Blocking One Histo (Nacalai Tesque). The slices were incubated overnight with an antibody against testosterone (1:50 dilution; Cloud-Clone Corp., TX, USA), at 4 °C. On the following day, the slices were rinsed in PBS and incubated with a secondary antibody conjugated with horseradish peroxidase. The slides were developed with diaminobenzidine (Vector Laboratories, Burlingame, CA, USA). Quantitative analysis of the histological staining was performed using the ImageJ software (National Institutes of Health, MD, USA). [...] A 50 mg/mL solution of 2,5-dihydroxybenzoic acid in a methanol: water mixture (7:3, v/v) served as a matrix. The matrix solution was sprayed uniformly over the slices by means of an airbrush with a 0.2 mm caliber nozzle (Procon Boy FWA Platinum; Mr. Hobby, Tokyo, Japan). MSI was performed using a MALDI TOF/TOF-type instrument (Autoflex) and the LTQ-XL linear ion trap mass spectrometer. Autoflex was used for imaging based on the MS data, and LTQ-XL was mainly used for imaging based on tandem mass spectrometry (MS/MS) data. This was done because Autoflex is suitable for simultaneous analysis of multiple samples, whereas LTQ-XL can perform simultaneous imaging on the basis of MS and MS/MS data. The data were acquired with a step size of 50 µm in both analyses. The FlexImaging software 4.0 (Bruker Daltonics) and ImageQuest software (Thermo Fisher Scientific) were used to create two-dimensional ion density maps. Normalization of the spectra to the total ion current was also performed in the imaging software. The distribution of the MS/MS product ions was visualized without normalization. […]

Pipeline specifications

Software tools ImageJ, ProCon, flexImaging, ImageQuest
Applications Miscellaneous, Mass spectrometry imaging, Microscopic phenotype analysis
Diseases Arthritis, Rheumatoid, Coronary Artery Disease, Diabetes Mellitus, Sleep Apnea, Obstructive, Pulmonary Disease, Chronic Obstructive
Chemicals Phosphatidylcholines, Testosterone, Eicosapentaenoic Acid